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Independent and combined effects of diethylhexyl phthalate and polychlorinated biphenyl 153 on sperm quality in the human and dog

Abstract

A temporal decline in human and dog sperm quality is thought to reflect a common environmental aetiology. This may reflect direct effects of seminal chemicals on sperm function and quality. Here we report the effects of diethylhexyl phthalate (DEHP) and polychlorinated biphenyl 153 (PCB153) on DNA fragmentation and motility in human and dog sperm. Human and dog semen was collected from registered donors (n = 9) and from stud dogs (n = 11) and incubated with PCB153 and DEHP, independently and combined, at 0x, 2x, 10x and 100x dog testis concentrations. A total of 16 treatments reflected a 4 × 4 factorial experimental design. Although exposure to DEHP and/or PCB153 alone increased DNA fragmentation and decreased motility, the scale of dose-related effects varied with the presence and relative concentrations of each chemical (DEHP.PCB interaction for: DNA fragmentation; human p < 0.001, dog p < 0.001; Motility; human p < 0.001, dog p < 0.05). In both human and dog sperm, progressive motility negatively correlated with DNA fragmentation regardless of chemical presence (Human: P < 0.0001, r = −0.36; dog P < 0.0001, r = −0.29). We conclude that DEHP and PCB153, at known tissue concentrations, induce similar effects on human and dog sperm supporting the contention of the dog as a sentinel species for human exposure.

Introduction

Over the last four decades, there has been increasing concern over declining human male reproductive health. Reduced sperm counts have been widely used as an index of male subfertility and meta-analytical studies indicate a 50% global reduction in quality from 1938 to 20111,2,3. Sperm morphology has also been reported to decrease over a period of 17 years in France with some geographical regional variation; Aquitaine and Midi-Pyrenees having the lowest morphology combined with the lowest concentrations4,5. These data are indicative of an environmental aetiology and, in support of this, epidemiological studies showing increased incidences of testicular cancer and malformations at birth have been linked to regions with reduced sperm counts6,7.

Temporal trends in human semen quality are paralleled by a similar trend in dogs that live in the human household, where sperm motility declined by 30% over a 26 year period8. In this latter study, all data was generated from a single laboratory using consistent techniques and thus did not suffer from changes in methodology and quality assurance over the time span encompassed in human meta-analytical studies9. These observations support the hypothesis that temporal trends in semen quality, both in the human and dog, are due to shared environmental factors and that the dog may be a sentinel for human exposure to such factors. Access to a controlled breeding population of assistance dogs that are routinely sampled for sperm quality provides a cost-effective means of sperm analyses without the stigma and social complications that accompany analogous human studies. Furthermore, there is considerable potential to extend these analyses to any individual or population of dogs. For example, although not investigated in the current study, this could be achieved by semen collection from the tail of the epididymis10 immediately after removal of dog testes at routine surgical neutering; a procedure that is performed on hundreds of thousands of dogs worldwide each year. In addition, semen collections from live dogs is a procedure that is tolerated by a majority of breeds11 unaccustomed to routine fertility monitoring and can therefore easily be carried out by a trained technician.

Declining sperm quality has been linked with the exposure to persistent anthropogenic chemicals, many of which exhibit endocrine disrupting activity6. Although the mechanisms underlying these putative effects are uncertain, historically, the period of fetal development has been highlighted as being particularly sensitive to chemicals with endocrine disrupting activity12. However, a number of studies have shown that environmental chemicals (ECs) are present in semen in a range of species, including the human, raising the possibility of a direct acute effect of chemicals on sperm13,14,15. In support of this theory, an elevated concentration of seminal bisphenol A (BPA) has been associated with infertility in men15,16 and elevated human seminal phthalate metabolites have been associated with reduced sperm counts17. In a separate study, the phthalates DEHP and di-n-butyl-phthalate (DBP) in human semen were reported to be inversely associated with motility and this was confirmed by the direct application of the same phthalates, at seminal concentrations, to sperm in vitro18. By contrast, PCB congeners 118, 126 and 153 were reported to have no negative effect on human sperm motility in vitro, both individually and in combination19. Similar findings have been reported in the dog where DEHP and PCB153, at concentrations detected in dog semen and testis, exhibited inhibitory and stimulatory effects respectively when tested on sperm motility in vitro8. In the same study, both DEHP and PCB153 were detected in a range of dry and wet dog foods indicative of a dietary source. Both DEHP and PCB153 are widely present in the environment and have been detected in tissues/fluids ranging from human breast milk to ovine liver. DEHP is a widely used plasticizer that leaches out into food and liquids and PCBs are lipophilic, and are therefore present in fatty foods20,21,22. Consequently, exposure occurs largely through the diet and these chemicals are deemed as risk factors for reproductive function23,24,25. In support of this, our own published study has shown that DEHP, PCB153 and other PCB congeners are present within both dry and wet dog food sources8 [DEHP: wet and dry food, 0.37 ± 0.10 and 0.20 ± 0.03 μg/g respectively; ∑PCBs: wet and dry food, 1.35 ± 0.5792 and 0.78 ± 0.223 μg/kg respectively; PCB153: wet and dry food, 0.39 ± 0.193 and 0.22 ± 0.11 μg/kg respectively].

Another parameter of ejaculate quality is the proportion of sperm that exhibit DNA fragmentation26. Environmental chemicals have been shown to induce both human and dog sperm DNA fragmentation8,27 and a commercial mixture of PCBs (Arochlor), administered to rats in vivo and added to sperm in vitro, also increased sperm DNA fragmentation28.

In total, these data suggest that environmental chemicals induce similar acute effects on human and dog sperm in vitro: measurement of sperm motility and DNA fragmentation are tried and tested measures of such chemical effects. Since sperm concentration would not alter during the period of in vitro culture and morphology is confounded by abnormality classification, partly due to swelling that may occur during culture and the generation of artefacts during processing, these parameters were not selected for testing acute chemical effects in vitro29,30. Notwithstanding, testing the effects of individual chemicals on sperm functional parameters does not represent “real-life” exposure to a mixture of chemicals, many of which exhibit synergistic, antagonistic or additive effects. Two chemicals known to be present in dog seminal plasma and testis were therefore selected and their effects tested both independently and in combination, on sperm motility and DNA fragmentation in both the human and dog.

Results

Chemical effects on percentage normal sperm motility in human and dog

The analyses of both the human and the dog sperm motility data found the interaction terms between PCB and DEHP to be significant (human p < 0.001; dog p < 0.05). This indicates that the dose response to either chemical was not independent of the level of the other chemical present. Figure 1 illustrates the effects of PCB153 and DEHP, individually and combined, on dog and human sperm motility. In dog sperm, PCB153 in the absence of DEHP induced a dose-dependent inhibitory effect on sperm motility (Fig. 1ai). In the presence of DEHP at 2x and 10x mean testis concentration (MTC), no dose dependent inhibitory effect of PCB153 was observed. However, reduced motility was observed in the presence of 100x DEHP co-incubated with 2x and 10x PCB153 (Fig. 1aii).

Effect of DEHP and PCB153 on dog and human sperm motility. Chemicals tested individually [ai,bi: PCB only; aii,bii: grey bars: DEHP only] and in combination [aii,bii]. Graphs display fixed concentrations of DEHP with increasing concentrations of PCB153 in dog [ai,aii: p <  0.01] and human [bi,bii: p <  0.001] sperm. Grade a motility: >25 μm/s Error bar = 1 Standard Error of Difference. MTC = Mean testis concentration.

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In contrast to the dog, neither PCB153 nor DEHP, in the absence of the other chemical, influenced human sperm motility (Fig. 1bi,bii). However, in the presence of 2x DEHP, a dose dependent inhibitory effect in response to PCB153 was observed (Fig. 1bii). The inhibitory effect of PCB153 was broadly maintained in the presence of 10x and 100x DEHP with the exception of 10x PCB153/10x DEHP and 100x PCB153/100x DEHP, where no inhibition was observed (Fig. 1bii).

Chemical effects on sperm DNA integrity in human and dog

For both the human and dog DNA fragmentation data, significant (P < 0.001) PCB.DEHP interaction terms were found. Again, this indicates that the response of DNA integrity to one chemical is not independent of the presence or level of the other chemical. Figure 2 illustrates the effects of PCB153 and DEHP, individually and combined, on both dog and human sperm DNA fragmentation using the sperm chromatin dispersion assay. In the dog, PCB153 in the absence of DEHP induced a dose-dependent increase in sperm DNA fragmentation (Fig. 2ai). A similar dose-dependent increase in DNA fragmentation was observed in response to DEHP in the absence of PCB153 (Fig. 2aii). When PCB153 and DEHP were tested in combination, DNA fragmentation was still increased at higher concentrations of PCB153 but the dose-dependent response was blunted (Fig. 2aii). The response of human sperm to PCB153 and DEHP independently and combined, paralleled that observed in the dog. Figure 2b illustrates that human sperm incubated with PCB153 or DEHP in the absence of the other chemical, exhibited a dose-dependent increase in DNA fragmentation (Fig. 2bi,bii). In addition, DNA fragmentation was increased in response to both chemicals tested in combination although additive effects were not apparent.

Effect of DEHP and PCB153 on dog and human sperm DNA fragmentation. Chemicals tested individually [ai,bi: PCB only; aii,bii: grey bars: DEHP only] and in combination [aii,bii]. Graphs display fixed concentrations of DEHP with increasing concentrations of PCB153 in dog [ai,aii: p ≤ 0.001] and human [bi,bii: p ≤ 0.001] sperm. Error bar = 1 Standard Error of difference between means. MTC = Mean testis concentration.

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Sperm DNA fragmentation and motility correlations

Figure 3 illustrates the relationship between progressive motility and DNA fragmentation in the presence and absence of each chemical independently and combined. Despite chemical effects on sperm motility (Fig. 1) and DNA fragmentation (Fig. 2), the relationship between these two parameters remained the same regardless of the nature of the chemical exposure. All correlations were significant except the human control samples [Dog (Fig. 3i, n = 352): Control; p < 0.05, r = −0.527, n = 22; DEHP; p < 0.05, r = −0.2862, n = 66; PCB-153; p < 0.01, r = −0.3276, n = 66; Mixture; p < 0.0001, r = −0.2826, n = 198; vs Human (Fig. 3ii, n = 288): Control; p > 0.05, r = −0.4374, n = 18; DEHP; p < 0.05, r = −0.3118, n = 54; PCB-153; p < 0.05, r = −0.2994, n = 54; Mixture; p < 0.0001, r = −0.4037, n = 162].

Correlation between progressive motility and DNA fragmentation in both dog and human sperm. Values from 32 sperm assessments (16 treatments, two time points). Each point represents a different sperm culture equating to a total n = 352 [dog] and n = 288 [human]. Colours denote culture media constituents to demonstrate spread: Control (black), DEHP (red), PCB153 (blue) and mixture (green). Dog (i, n = 352): Control; p < 0.05, r = −0.527, n = 22; DEHP; p < 0.05, r = −0.2862, n = 66; PCB153; p < 0.01, r = −0.3276, n = 66; Mixture; p < 0.0001, r = −0.2826, n = 198; vs Human (ii, n = 288): Control; p > 0.05, r = −0.4374, n = 18; DEHP; p < 0.05, r = −0.3118, n = 54; PCB153; p < 0.05, r = −0.2994, n = 54; Mixture; p < 0.0001, r = −0.4037, n = 162]. Confidence bands (95%) plotted for visual purposes only. Progressive motility based on WHO pre-2010 where sperm swimming grades a and b are combined: ≥5 µm/s.

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Discussion

Data presented in this paper are significant because we conclusively demonstrate that a selected phthalate (DEHP) and PCB congener (PCB153), at concentrations relevant to environmental exposure, reduce dog sperm motility and increase DNA fragmentation in vitro. In the human, our data showing reduced motility and increased DNA fragmentation are indicative of similar sensitivities to these chemicals in both species. Furthermore, when the chemicals were combined and co-incubated with dog or human sperm, the overall impact on motility or DNA fragmentation was dependent on the concentration ratio. This is the first study to select two environmental chemicals and to test them at four concentrations in all possible combinations representative of those found in the male reproductive tract and fluids. In addition, a negative correlation between motility and DNA fragmentation, that incorporates chemical variables, is described in both species.

Our data on the sensitivity of sperm to short term chemical exposure support similar studies using a range of environmentally relevant chemical challenges in vitro. For example, DEHP, DBP and mono-n-butyl phthalate (MBP), are reported to reduce sperm motility in vitro when added at seminal concentrations measured in infertile men18,31. A mixture of PCBs are also reported to reduce sperm motility in both the human and pig32,33 and in the human, p,p’-dichloro-diphenyl-dichloro-ethylene (p,p’-DDE) has been shown to increase Ca2+ uptake by sperm in a mechanism that involves the CatSper channel34.

To minimise the effects of pre-exposure to chemicals present in ejaculates collected for culture, the current study used a concentration range that encompassed the reported variability in seminal concentrations and reduced pre-existing chemical concentrations by sperm processing and washing prior to culture. The subsequent comparison to controls, with no further chemical added, provided a means of testing chemical effects. Despite these steps, pre-exposure differences will inevitably exist between, and within, species. For example, in the current study, when DEHP is present at twice the concentration found in testis, dog sperm appears to be less sensitive to lower PCB concentrations than human sperm. In populations of men from Greenland, Sweden, Poland and the Ukraine, increased seminal PCB153 has been consistently associated with reduced sperm motility but not concentration or morphology35,36. In addition, sperm DNA fragmentation was positively associated with PCB153 in European populations but not in samples from Greenlandic men: an observation that likely reflects different pre-exposures.

In the current study, our intention was not to measure and equate chemical concentrations in each individual sample with sperm motility and fragmentation, but to use a concentration range previously established in the dog as a ballpark estimate of chemical concentrations in the male reproductive tract8. That is, concentrations found in the dog reproductive tract and seminal plasma, used as an indicator of those likely present in the human. In support of this contention, human seminal PCB (total) and DEHP concentrations reported in populations of fertile men [total PCB: up to 5.8 ng/ml, DEHP: mean of 0.61 μg/ml]37,38 are comparable to those detected in the dog [PCB: 0.26–13.2 ng/ml; DEHP: 0.75–37.5 μg/ml]8.

Notably, elevated concentrations of both chemicals have been linked with reduced human male fertility18. Semen PCB concentrations have been reported to be higher in a population of ‘infertile’ men (parameters stated as <20 million/ml or <25% progressive motility and/or <30% normal morphology)25. Further, a research group in China reported an association between increased urinary phthalate metabolites, reduced sperm count and increased sperm DNA damage14. Although the mechanism was not proven, the authors suggest that this likely reflects chemical effects on testicular Sertoli and germ cells as reported in animal studies39,40,41,42.

Although both DEHP and PCB153 have been reported to reduce sperm motility and increase sperm DNA fragmentation individually in the human8,18,37, they have not been assessed in combination, at environmentally relevant concentrations, as reported here. A limited number of studies have investigated some combinations of environmental chemicals for their effect on human sperm function32. Real-life exposure is to a complex mixture of chemicals that are likely to exhibit synergistic or antagonistic, as well as additive effects, on sperm. Mixtures of endocrine disrupting chemicals have been shown to cooperatively increase Ca2+ concentrations in sperm through the activation of the principle calcium channel CatSper43.

In the current study, the mechanisms that underlie the concentration ratio-dependent effects of the two chemicals combined remain uncertain. PCBs and DEHP are considered to be pro-estrogenic and anti-androgenic respectively raising the possibility that a change in concentration ratio may alter the relative activation of sperm estrogen or androgen receptors44,45. Indeed, in a population of ‘infertile’ men (parameters stated as sperm count < 20 × 106, motility < 50%, morphology < 14% normal) androgen receptor expression is reported to positively correlate with sperm motility46 and PCBs are reported to affect sperm concentration and motility relative to the number of CAG repeats in the androgen receptor gene47. This may account for PCB influences on motility even in the presence of DEHP. Estrogenic compounds have also been reported to reduce human sperm motility in vitro via an induction in redox activity and this mechanism has also been linked to the induction of DNA fragmentation by 2-hydroxy estradiol48.

The greater consistency of chemical effects on human and dog sperm DNA fragmentation compared to motility is interesting and emphasises the importance of looking at more than one sperm functional/viability parameter when assessing environmental effects. It is important to note however that despite this subtle difference, the two sperm parameters were highly correlated in both species and remained so in the presence of the chemicals independently and combined. This is an important observation since it has been reported that human male infertility is associated with increased levels of sperm DNA damage and that sperm motility defects are highest in samples with increased DNA fragmentation49,50. This raises the possibility that there may be a similar relationship between sperm DNA fragmentation, motility and fertility in the dog.

In conclusion, we have demonstrated that the use of low dose tissue relevant concentrations of DEHP and PCB153, independently and in combination, negatively impact on sperm motility and DNA fragmentation in samples obtained from humans and dogs. Since these effects are broadly similar in both species, this raises the possibility that the environmental impact of chemicals in the dog may provide a means of investigating pollutant effects on mammalian fertility in a species in which external influences, such as diet, are better controlled than in an equivalent human study.

Methods

Ethical Approval

Human

Semen donations were obtained from anonymous HFEA registered donors (n = 9) attending the fertility unit at Nottingham University Hospitals. Donors provided informed consent for the use of samples in this research project ensuring ‘General Data Protection Regulation’ compliance. Each donor was initially screened following HFEA and British Fertility Society protocols51. No samples from fertility patients were used and all donors completed HFEA consent forms. Ethical approval was obtained from the School of Veterinary Medicine Ethical Review Committee [Reference 1511,150723]. The HFEA consent forms completed by donors and ethical approval documents were also approved by the Chair of the Ethical Review Committee of the Faculty of Medicine and Health Sciences, University of Nottingham. In accordance with the Royal College of Pathologists guidance on the use of pathological specimens [https://www.rcpath.org/resourceLibrary/the-retention-and-storage-of-pathological-records-and-specimens-5th-edition-.html], no further ethical approval was required.

Dog

Semen was collected as part of routine reproductive examination of stud dogs subject to owner consent with full GDPR compliance. Due to dog sperm being collected as part of routine reproductive health checks, the economical outlay associated with sample collection was minimised. The dogs resided in the same region of the UK and lived in the modern household with owners briefed on use of controlled diet and exercise regimes. All semen collections were performed in accordance with relevant guidelines and regulations and the collection protocol was approved by the School of Veterinary Medicine Ethical Review Committee (Refs: 208 101012, 513 120117 and 1097 140227).

Human sperm collection and preparation

Samples were liquefied for 20 minutes at room temperature prior to semen preparation. Ejaculate underwent density gradient centrifugation using a Universal 320 R Hettich centrifuge (DJB Labcare, Newport Pagnell, UK) at 25 °C. Briefly, one millilitre of 40% isotonic density gradient was loaded onto one millilitre of 80% isotonic medium [PureSperm 40/80, Nidacon, Sweden]. Two millilitres of liquefied semen were loaded onto both isotonic mediums and reagents centrifuged at 300 × g for 22 minutes. On completion, the sperm rich pellet was re-suspended using 1.5 ml PureSperm wash (pH range of 7.3–8.5; osmolality: 290–300 mOsm/kg H2O, Nidacon, Sweden).

Dog sperm collection

Ejaculate was collected from stud dogs (n = 11) by routine digital manipulation. The sperm rich fraction (fraction 2) was collected into sterile plastic 15 millilitre Greiner centrifuge tubes [Sigma-Aldrich, Dorset, UK]. INRA extending medium [INRA, Nouzilly, France] was added to sperm at a 2:1 ratio to aid sperm survival.

Chemical preparation

The two chemicals selected for co-culture with sperm were diethylhexyl phthalate (DEHP) and polychlorinated biphenyl congener 153 (PCB153) and concentrations were calculated relative to those present in dog testicular tissue8. The rationale for this was (1) dog testis concentrations of both chemicals have been established and standardised in our previous study and are reflective of exposure of the male reproductive tract8 (2) dog and human semen PCB concentrations are variable but generally higher than testis and in range of the concentrations tested in vitro8,25,52 (3) although dog semen DEHP concentrations have not been determined, due to the large amount of dry material required, reported human DEHP concentrations are in range of established dog testis measurements14,18,53. On this basis, dog testis concentrations were used as a standardised measure of environmental exposure relevant to both species. DEHP (CAS no: 117-81-7) and PCB153 (CAS no: 35065-27-1) [Sigma-Aldrich, Dorset, U.K.] were dissolved in 100% dimethylsulphoxide (DMSO) and diluted with PBS to 4x, 20x and 200x mean testis concentration containing 0.02% DMSO. Chemical preparations were co-incubated with sperm at a 1:1 ratio with a final exposure concentration of 2x, 10x and 100x mean testis concentration. Sperm were incubated with each chemical at each concentration individually and with a mixture of the two chemicals in all 16 combination ratios (Table 1). Acute treatment effects were assessed at 10 minutes and 3 hours and control incubations carried out with 0.01% DMSO only.

Full size table

Motility assessment

Sperm motility was assessed using Computer Assisted Sperm Analysis (CASA) software. Sperm were acclimatised for two minutes on a 37 °C stage prior to assessment. Five µl of sperm, irrespective of species, were pipetted into a specialised 20 µl cellvision glass slide counting chamber (Code CV1020-2CV; CellVision, the Netherlands). A minimum of 200 sperm were assessed for each treatment. For human sperm, motility was tracked by use of the diagnostic software ‘SAMi’ (Procreative Diagnostics Ltd, Staffordshire, UK) and an Olympus BH2 Microscope [KeyMed (Medical and Industrial Equipment) Ltd, Essex, UK]. Dog sperm motility was assessed using the Hobson’s CASA tracking system (Hobson’s tracking systems Ltd., Sheffield, UK) and viewed using a negative-high phase contrast objective (x20) on an Olympus BH2 microscope fitted with a camera ocular. For both species, sperm motility was assessed according to WHO 199954 where grade a motility was classified as ≥ 25 µm/s and progressive motility ≥ 5 µm/s (grades a & b combined).

DNA fragmentation

Sperm DNA fragmentation was quantified by defragmentation index (sDFI %) measured using the sperm chromatin dispersion assay55. Sperm were assessed based on the size of the halos, indicative of sperm that exhibited nuclei with non-fragmented DNA. Sperm with denatured or fragmented DNA were identified by the absence of a halo, or a halo that was exceedingly small. Briefly, equal volumes of intact, unfixed sperm and 1% agarose solution were combined. Fifteen microliter aliquots of sperm-agarose suspensions were pipetted onto agarose pre-coated poly-L-lysine slides (CAS: P4981; ThermoFisher Scientific Ltd. UK), covered with a 22 mm × 22 mm cover-slip, and placed in a 4 °C environment for five minutes to fix the sample. After an acid treatment (0.08 M HCl) of seven minutes, sperm were incubated in a lysing reagent (0.8 M DTT, 0.4 M Tris, 2 M NaCl, 1% triton-X) for 20 minutes. Slides were then submerged in dH2O for a period of five minutes followed by dehydration through a series of ethanol solutions (70%, 90% and 100% respectively). Visualisation was obtained using Diff-Quick staining reagents (eosinophilic and basophilic stains; CAS 9990700, ThermoFisher Scientific Ltd, Paisley, UK) and analysis undertaken using oil immersion, at 1000x magnification [Leica DM 5000 B microscope; Leica Microsystems, Milton Keynes, UK]. A minimum of 200 sperm were assessed for each treatment. Positive controls were incubated with 300 µm H2O2 before denaturation and negative controls by omission of the denaturation step. To determine the defragmentation index, the number of fragmented sperm was divided by the total number of sperm counted. This provided a value for the proportion of sperm that were fragmented. This proportion was then plotted on the logit scale.

Experimental design and Statistical analysis

For both the human and dog experiments, sperm samples were treated with PCB153 at one of four concentrations in combination with DEHP, also at one of four concentrations. The concentrations used for both chemicals corresponded to 0x, 2x, 10x and 100x the baseline concentration measured from dog testes. Thus there were 16 possible combinations of PCB153 and DEHP. Each combination was randomly allocated to one of 16 separate sub-samples of sperm, from each replicate donor.

Statistical analysis was undertaken using GenStat 17th edition (VSN International Ltd, Hempstead, UK). The proportions out of known numbers of sperm that had a particular characteristic were analysed as grouped binary data by fitting a generalised linear mixed model with a logit link function, assuming a binomial error distribution. The fixed effects included in the statistical model were PCB, DEHP and the PCB.DEHP interaction term. Another fixed effect, TIME, and its interaction terms were included when two repeated samples from the same tube were measured three hours apart. The random effects were donor and culture-tube within donor.

The statistical significance of a fixed effect was tested using an F-Ratio test. The analysis output provided predicted mean proportions and standard errors of difference between means which were graphically represented on a logit scale. Logit values were converted back into proportions and graphically plotted [GraphPad Prism 7.0, GraphPad Ltd, California, USA]. A single error bar on each figure represents the standard error of the difference between means. Where correlations were investigated, data was assumed to be non-normally distributed and a Spearman’s rank correlation analysis was undertaken to provide correlation coefficients between motility and DNA fragmentation.

Data Availability

The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

References

  1. 1.

    Carlsen, E., Giwercman, A., Keiding, N. & Skakkebaek, N. E. Evidence for decreasing quality of semen during past 50 years. BMJ305, 609–613 (1992).

    CASArticle Google Scholar

  2. 2.

    Swan, S. H., Elkin, E. P. & Fenster, L. The question of declining sperm density revisited: an analysis of 101 studies published 1934–1996. Environ. Health Perspect.108, 961–966 (2000).

    CASArticle Google Scholar

  3. 3.

    Levine, H. et al. Temporal trends in sperm count: a systematic review and meta-regression analysis. Hum. Reprod. Update23, 646–659 (2017).

    Article Google Scholar

  4. 4.

    Rolland, M., Le Moal, J., Wagner, V., Royere, D. & De Mouzon, J. Decline in semen concentration and morphology in a sample of 26,609 men close to general population between 1989 and 2005 in France. Hum. Reprod. 28, 462–470 (2013).

  5. 5.

    Le Moal, J. et al. Semen quality trends in French regions are consistent with a global change in environmental exposure. Reproduction147, 567–574 (2014).

    Article Google Scholar

  6. 6.

    Bay, K., Asklund, C., Skakkebaek, N. E. & Andersson, A.-M. Testicular dysgenesis syndrome: possible role of endocrine disrupters. Best Pract. Res. Clin. Endocrinol. Metab.20, 77–90 (2006).

    CASArticle Google Scholar

  7. 7.

    Skakkebaek, N. E. Sperm counts, testicular cancers, and the environment. BMJ (Clinical research ed.)359, j4517 (2017).

    Article Google Scholar

  8. 8.

    Lea, R. G. et al. Environmental chemicals impact dog semen quality in vitro and may be associated with a temporal decline in sperm motility and increased cryptorchidism. Sci. Rep.6, 31281 (2016).

    ADSCASArticle Google Scholar

  9. 9.

    Pacey, A. A. Are sperm counts declining? Or did we just change our spectacles? Asian J. Androl.15, 187–190 (2013).

    Article Google Scholar

  10. 10.

    Ponglowhapan, S., Chatdarong, K., Sirivaidyapong, S. & Lohachit, C. Freezing of epididymal spermatozoa from dogs after cool storage for 2 or 4 days. Theriogenology66, 1633–1636 (2006).

    CASArticle Google Scholar

  11. 11.

    Hermansson, U. & Linde Forsberg, C. Freezing of stored, chilled dog spermatozoa. Theriogenology65, 584–593 (2006).

    Article Google Scholar

  12. 12.

    Lea, R. G. et al. Endocrine disruptors and ovine reproductive development. In Reproduction in Domestic Ruminants Vol.VIII (eds Juengel, J. et al.) 209–227 (Context Products Ltd., UK, 2014).

  13. 13.

    Kamarianos, A., Karamanlis, X., Theodosiadou, E., Goulas, P. & Smokovitis, A. The presence of environmental pollutants in the semen of farm animals (bull, ram, goat, and boar). Reprod. Toxicol.17, 439–445 (2003).

    CASArticle Google Scholar

  14. 14.

    Wang, Y.-X. et al. Phthalate exposure and human semen quality: Results from an infertility clinic in China. Environ. Res.142, 1–9 (2015).

    ADSCASArticle Google Scholar

  15. 15.

    La Rocca, C. et al. Exposure to Endocrine Disruptors and Nuclear Receptors Gene Expression in Infertile and Fertile Men from Italian Areas with Different Environmental Features. Int. J. Environ. Res. Public Health12, 12426–12445 (2015).

    Article Google Scholar

  16. 16.

    Vitku, J. et al. Difflerences in bisphenol A and estrogen levels in the plasma and seminal plasma of men with different degrees of infertility. Physiol. Res.64(Suppl 2), S303–11 (2015).

    CASPubMed Google Scholar

  17. 17.

    Chang, W.-H., Wu, M.-H., Pan, H.-A., Guo, P.-L. & Lee, C.-C. Semen quality and insulin-like factor 3: Associations with urinary and seminal levels of phthalate metabolites in adult males. Chemosphere173, 594–602 (2017).

    ADSCASArticle Google Scholar

  18. 18.

    Pant, N. et al. Environmental and experimental exposure of phthalate esters: the toxicological consequence on human sperm. Hum. Exp. Toxicol.30, 507–514 (2011).

    CASArticle Google Scholar

  19. 19.

    Pflieger-Bruss, S. et al. Effects of single non-ortho, mono-ortho, and di-ortho chlorinated biphenyls on human sperm functions in vitro. Reprod. Toxicol.21, 280–284 (2006).

    CASArticle Google Scholar

  20. 20.

    Kastner, J., Cooper, D. G., Maric, M., Dodd, P. & Yargeau, V. Aqueous leaching of di-2-ethylhexyl phthalate and ‘green’ plasticizers from poly(vinyl chloride). Sci. Total Environ.432, 357–364 (2012).

    ADSCASArticle Google Scholar

  21. 21.

    Erythropel, H. C., Maric, M., Nicell, J. A., Leask, R. L. & Yargeau, V. Leaching of the plasticizer di(2-ethylhexyl)phthalate (DEHP) from plastic containers and the question of human exposure. Appl. Microbiol. Biotechnol.98, 9967–9981 (2014).

    CASArticle Google Scholar

  22. 22.

    Faroon, O. & Ruiz, P. Polychlorinated biphenyls: New evidence from the last decade. Toxicol. Ind. Health32, 1825–1847 (2016).

    CASArticle Google Scholar

  23. 23.

    Pocar, P. et al. Exposure to di(2-ethyl-hexyl) phthalate (DEHP) in utero and during lactation causes long-term pituitary-gonadal axis disruption in male and female mouse offspring. Endocrinology153, 937–948 (2012).

    CASArticle Google Scholar

  24. 24.

    Oskam, I. C. et al. Effects of long-term maternal exposure to low doses of PCB126 and PCB153 on the reproductive system and related hormones of young male goats. Reproduction130, 731–742 (2005).

    CASArticle Google Scholar

  25. 25.

    Rozati, R., Reddy, P. P., Reddanna, P. & Mujtaba, R. Role of environmental estrogens in the deterioration of male factor fertility. Fertil. Steril.78, 1187–1194 (2002).

    Article Google Scholar

  26. 26.

    Rex, A. S., Aagaard, J. & Fedder, J. DNA fragmentation in spermatozoa: a historical review. Andrology5, 622–630 (2017).

    CASArticle Google Scholar

  27. 27.

    Evenson, D. P. & Wixon, R. Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA). Toxicol. Appl. Pharmacol.207, 532–537 (2005).

    Article Google Scholar

  28. 28.

    Aly, H. A. A. Aroclor 1254 induced oxidative stress and mitochondria mediated apoptosis in adult rat sperm in vitro. Environ. Toxicol. Pharmacol.36, 274–283 (2013).

    CASArticle Google Scholar

  29. 29.

    Mortimer, D. & Menkveld, R. Sperm morphology assessment—historical perspectives and current opinions. J. Androl.22, 192–205 (2001).

    CASPubMed Google Scholar

  30. 30.

    Boersma, A., Rasshofer, R. & Stolla, R. Influence of sample preparation, staining procedure and analysis conditions on bull sperm head morphometry using the morphology analyser integrated visual optical system. Reprod. Domest. Anim.36, 222–229 (2001).

    CASArticle Google Scholar

  31. 31.

    Xie, F. et al. Effects of two environmental endocrine disruptors di-n-butyl phthalate (DBP) and mono-n-butyl phthalate (MBP) on human sperm functions in vitro. Reprod. Toxicol.83, 1–7 (2019).

    CASArticle Google Scholar

  32. 32.

    Jiang, L.-G. et al. Toxic effects of polychlorinated biphenyls (Aroclor 1254) on human sperm motility. Asian J. Androl.19, 561–566 (2017).

    CASArticle Google Scholar

  33. 33.

    Campagna, C., Guillemette, C., Ayotte, P. & Bailey, J. L. Effects of an environmentally relevant organochlorine mixture and a metabolized extract of this mixture on porcine sperm parameters in vitro. J. Androl.30, 317–324 (2009).

    CASArticle Google Scholar

  34. 34.

    Tavares, R. S., Escada-Rebelo, S., Correia, M., Mota, P. C. & Ramalho-Santos, J. The non-genomic effects of endocrine-disrupting chemicals on mammalian sperm. Reproduction151, R1–R13 (2016).

    CASArticle Google Scholar

  35. 35.

    Toft, G. Persistent organochlorine pollutants and human reproductive health. Dan. Med. J.61, B4967 (2014).

    PubMed Google Scholar

  36. 36.

    Toft, G. et al. Semen quality and exposure to persistent organochlorine pollutants. Epidemiology17, 450–458 (2006).

    Article Google Scholar

  37. 37.

    Bush, B., Bennett, A. H. & Snow, J. T. Polychlorobiphenyl congeners, p,p’-DDE, and sperm function in humans. Arch. Environ. Contam. Toxicol.15, 333–341 (1986).

    CASArticle Google Scholar

  38. 38.

    Han, S. W. et al. An exposure assessment of di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) in human semen. J. Toxicol. Environ. Heal. Part A72, 1463–1469 (2009).

    CASArticle Google Scholar

  39. 39.

    Jones, S., Boisvert, A., Francois, S., Zhang, L. & Culty, M. In utero exposure to di-(2-ethylhexyl) phthalate induces testicular effects in neonatal rats that are antagonized by genistein cotreatment. Biol. Reprod.93(92), 1–14 (2015).

    CAS Google Scholar

  40. 40.

    Richburg, J. H. & Boekelheide, K. Mono-(2-ethylhexyl) phthalate rapidly alters both Sertoli cell vimentin filaments and germ cell apoptosis in young rat testes. Toxicol. Appl. Pharmacol.137, 42–50 (1996).

    CASArticle Google Scholar

  41. 41.

    Ungewitter, E. et al. From the Cover: Teratogenic Effects of in Utero Exposure to Di-(2-Ethylhexyl)-Phthalate (DEHP) in B6:129S4 Mice. Toxicol. Sci.157, 8–19 (2017).

    CASPubMedPubMed Central Google Scholar

  42. 42.

    Zhang, T. et al. Melatonin protects prepuberal testis from deleterious effects of bisphenol A or diethylhexyl phthalate by preserving H3K9 methylation. J. Pineal Res.65, e12497 (2018).

    Article Google Scholar

  43. 43.

    Schiffer, C. et al. Direct action of endocrine disrupting chemicals on human sperm. EMBO Rep.15, 758–765 (2014).

    CASArticle Google Scholar

  44. 44.

    Aquila, S. et al. Estrogen receptor (ER)alpha and ER beta are both expressed in human ejaculated spermatozoa: evidence of their direct interaction with phosphatidylinositol-3-OH kinase/Akt pathway. J. Clin. Endocrinol. Metab.89, 1443–1451 (2004).

    CASArticle Google Scholar

  45. 45.

    Aquila, S. et al. Human sperm express a functional androgen receptor: effects on PI3K/AKT pathway. Hum. Reprod.22, 2594–2605 (2007).

    CASArticle Google Scholar

  46. 46.

    Zalata, A. A. et al. Androgen receptor expression relationship with semen variables in infertile men with varicocele. J. Urol.189, 2243–2247 (2013).

    CASArticle Google Scholar

  47. 47.

    Giwercman, A., Rylander, L. & Lundberg Giwercman, Y. Influence of endocrine disruptors on human male fertility. Reprod. Biomed. Online15, 633–642 (2007).

    CASArticle Google Scholar

  48. 48.

    Bennetts, L. E. et al. Impact of estrogenic compounds on DNA integrity in human spermatozoa: evidence for cross-linking and redox cycling activities. Mutat. Res.641, 1–11 (2008).

    ADSCASArticle Google Scholar

  49. 49.

    Velez de la Calle, J. F. et al. Sperm deoxyribonucleic acid fragmentation as assessed by the sperm chromatin dispersion test in assisted reproductive technology programs: results of a large prospective multicenter study. Fertil. Steril.90, 1792–1799 (2008).

    Article Google Scholar

  50. 50.

    Belloc, S. et al. Sperm deoxyribonucleic acid damage in normozoospermic men is related to age and sperm progressive motility. Fertil. Steril.101, 1588–1593 (2014).

    CASArticle Google Scholar

  51. 51.

    Hamilton, M. et al. Working Party on Sperm Donation Services in the UK. Hum. Fertil.11, 147–158 (2008).

    Article Google Scholar

  52. 52.

    Dallinga, J. W. et al. Decreased human semen quality and organochlorine compounds in blood. Hum. Reprod.17, 1973–1979 (2002).

    CASArticle Google Scholar

  53. 53.

    Zhang, Y.-H., Zheng, L.-X. & Chen, B.-H. Phthalate exposure and human semen quality in Shanghai: a cross-sectional study. Biomed. Environ. Sci.19, 205–209 (2006).

    CASPubMed Google Scholar

  54. 54.

    WHO. Laboratory Manual for the examination of human semen and sperm – cervical mucus interaction. (Cambridge University Press, Cambridge, 1999).

  55. 55.

    Fernandez, J. L. et al. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil. Steril.84, 833–842 (2005).

    CASArticle Google Scholar

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Acknowledgements

We thank Karen Pooley for the management of the human donors attending the Fertility Unit at Nottingham University Hospital and for expert technical advice and assistance. We also thank Natasha White for the collection of ejaculates from a population of assistance dogs. The work was supported by University of Nottingham internal funds.

Author information

Author notes
  1. Rebecca N. Sumner

    Present address: Hartpury University, Gloucester, GL19 3BE, UK

Affiliations

  1. School of Veterinary Medicine & Science, University of Nottingham, Sutton Bonington, LE12 5RD, UK

    Rebecca N. Sumner, Gary C. W. England & Richard G. Lea

  2. Fertility Unit, East Block B floor, Nottingham University Hospital, Nottingham, NG7 2UH, UK

    Mathew Tomlinson

  3. School of Biosciences, University of Nottingham, Sutton Bonington, LE12 5RD, UK

    Jim Craigon

Contributions

R.G.L. and R.N.S. conceived of and designed the study. M.T. managed the human donors and collection of human semen samples and G.C.W.E. managed the collection of canine samples. All authors provided academic input into the paper. R.N.S. carried out the computer assisted sperm analysis, chromatin dispersion assay and subsequent analysis. J.C. provided expert statistical advice and contributed to the analysis. R.G.L. and R.N.S. wrote the paper.

Corresponding author

Correspondence to Richard G. Lea.

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Competing Interests

The authors declare no competing interests.

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Sumner, R.N., Tomlinson, M., Craigon, J. et al. Independent and combined effects of diethylhexyl phthalate and polychlorinated biphenyl 153 on sperm quality in the human and dog. Sci Rep9, 3409 (2019). https://doi.org/10.1038/s41598-019-39913-9

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Collect and Freeze Semen

OUR FREEZING CENTER WILL BE CLOSING AT THE END OF 2021. ALL SEMEN WILL BE SENT TO ZOETIS OR NC STATE VET SCHOOL FOR LONG TERM STORARGE. 

WE ARE CURRENTLY NOT DOING FREEZES, DUE TO THE PANDEMIC. HOWEVER YOU CAN CONTACT NC STATE VET SCHOOL FOR INFORMATION ABOUT THEIR FREEZING CENTER CALL  (919) 513 6300 option 2 or  email to [email protected]

We are a freezing center for Zoetis. We do store semen at our facility. If you are going to be using us in the near future for your dog's reproductive needs, you can go ahead and have semen shipped to us. However please let us know that you are having semen shipped prior to shipping it to us. 


Please contact us for the Collect and Freeze requirements and process. Each collection has to be scheduled in advance, and collections are only done first thing in the mornings. The process takes about 6 hours, even though the collected dog is only in the office for about 30 minutes. 

Collect & Freeze Information: 

Q. What do I need to do to collect semen on my stud dog?

A. That's an easy one! Just call our office at the number listed and we can make you an appointment. Collections for freezing must be done at 9am, no later. It takes 6 hours to take semen thru the freezing process; however the dog is only here for about 20-30 minutes.


Q. What will I need to bring to my appointment?

A. You will need to bring the following:

  1. You are required to bring your AKC or other registration papers verifying that you are a current owner of the stud. (for unregistered dogs this does not apply)
  2. You will need a copy of your DNA Profile, or we can collect the DNA sample and send it in for an additional fee.
  3. You will need a current Brucellosis test.
  4. A semen freezing contract must be completed, since we are a freezing center for Zoetis.

Q. What if I want to send my stud with an agent for collection?

A. Please provide that person with all the items above and payment. Please note that the semen contract MUST be signed by the STUD DOG OWNER. The semen will be collected and stored in Stud owners' name.


Q. Can I let someone else collect my stud and let them own the semen?

A. Yes and No. Semen Ownership can be transferred to persons, not listed as an owner on the registration papers. The semen will need to first be registered to the stud dog owner and then transferred to the new owner.


Q. Is there a cost for annual semen storage?

A. Yes, the annual storage fees are ~$120.00 per year.


Q. How do I arrange to ship my frozen semen?

A. Frozen semen shipping should be planned ahead of time. Please remember that there are a limited number of shipping tanks available for use. It takes 24 hours to charge a shipping dewar. Extra fees do apply for STAT charging and weekend/holiday deliveries.

Источник: https://plantationanimalhospitalnc.com/collect-and-freeze-semen

Animal Breeding and Reproductive Services
in Frederick County

Consistent winner of Frederick's Best Vet, 2007-2021!

Buckeystown Veterinary Hospital offers reproductive services for both companion and farm animal species.

The following reproductive services are offered for the species listed below. If you require a breeding or reproductive service and do not see it listed, contact us for a consultation at:

Breeding & Reproduction Services for Dogs

  • Pre-breeding consultation and evaluation
  • Brucellosis testing
  • Progesterone testing
  • Vaginal cytology
  • Breeding management
  • Artificial insemination
  • Surgical insemination
  • Pregnancy diagnosis by ultrasound
  • “Puppy count” X-rays
  • Dystocia management
  • Caesarian section
  • Neonatal care
  • Semen collection and evaluation
  • Semen collection and shipment
  • Semen collection, freezing, and storage

Breeding & Reproductive Services for Horses

  • Pre-breeding consultation and examination
  • Breeding soundness examination
  • Breeding management
  • Artificial insemination using fresh, chilled, or frozen semen
  • Pregnancy diagnosis by ultrasound
  • Twin management
  • Late-term mare evaluation
  • Neonatal care

Breeding & Reproductive Services for Cattle

  • Breeding management
  • Pregnancy diagnosis by palpation or blood test
  • Neonatal care

Breeding & Reproductive Services for Small Ruminants

  • Breeding management
  • Pregnancy diagnosis by ultrasound
  • Neonatal care

Breeding & Reproductive Services for Llamas & Alpacas

  • Breeding management
  • Evaluation of problem breeders
  • Pregnancy diagnosis by palpation and ultrasound
  • Neonatal care
Источник: https://www.buckeystownvet.com/veterinary-services/breeding-reproduction.html

Semen collection in the dog

This review will discuss semen collection in the dog. Semen samples may be collected from male dogs for the purposes of artificial insemination, cryopreservation or diagnosis. The materials needed for semen collection depend on which method is used and the collector's level of expertise with this procedure. At minimum, two sterile centrifuge tubes or specimen cups can be used to collect semen as it is ejaculated (for the combined first and second fractions and for the third fraction). The most common method for semen collection in the dog is by digital stimulation. Under ideal conditions, this procedure is performed in the presence of an estrous bitch. Initially, the dog's penis is vigorously massaged through the prepuce at the level of the bulbus glandis (caudal-most aspect of the prepuce) until a partial erection develops (initial engorgement of the bulbus glandis). The prepuce is quickly retracted past the bulbus glandis and firm constant pressure is applied to the penis behind the bulbus glandis by squeezing the penis between index finger and thumb. Pelvic thrusting may occur following application of pressure behind the bulbus glandis during the development a "full" erection. The ejaculate is composed of three fractions: first (sperm-poor), second (sperm-rich) and third (prostatic fluid). In addition to digital stimulation of the penis, spermatozoa have been collected from dogs using electroejaculation and pharmacologic methods.

Источник: https://pubmed.ncbi.nlm.nih.gov/15993482/

More Info

We are proud to be an AKC approved Freezing Center.  Here are some of the frequently asked questions regarding canine semen freezing:

1)   Why should I freeze my dog’s Semen?

Long Term Storage: To ensure breeding availability for future generations, you should have his semen frozen if your stud has qualities which are valuable.

For Breeding When the Stud is Not Available: Live breeding can be limited by the stud’s show or trial schedule, overbooking for the stud’s service or other scheduling conflicts.  The use of frozen semen allows availability during the bitch’s fertile time.

Long-Distance and International Breeding: Long-distance breedings may be accomplished using semen which has been either fresh-extended or frozen and eliminates the need to transport the bitch or stud dog.

2)  How is Semen collected?

Semen is collected from the stud dog by manual stimulation; the different parts or fractions of the ejaculation are collected separately, so that good quality sperm-rich semen is frozen and stored. In general, semen of better quality with higher sperm count is collected when the dog’s libido is high.  Therefore, try to closely approximate a typical breeding situation for each stud: owners are encouraged to provide a bitch in season to use as a “teaser”.  In addition, if the dog associates a particular item with breeding, such as a rug, table, breeding rack, etc., that item should be brought to the collections.

3)  What kind of paper work is needed to collect my stud?   

  • A copy of the stud’s individual registration papers.
  • Positive identification such as a tattoo or microchip.
  • Completed Semen Freeze Agreement which can be emailed to you.
  • Completed Semen Freeze Authorization which can be emailed to you.
  • A copy of the DNA Profile for your dog (which is required by AKC).  If you do not have a DNA Profile on your dog, we will submit a cheek swab sample to the AKC.
  • A copy of a negative Brucellosis test.  If your stud has not had a Brucellosis test performed then we can have the test performed the same day as the collect and freeze.  This test is a MUST in order for your males semen to be stored in our liquid nitrogen tanks.

4)  What does it cost to freeze semen?

Semen freezing is a multi-step process utilizing a variety of equipment and materials.  Please see our Reproduction price sheet in the forms section of our website for detailed information.

5)  How do I set up an appointment for Semen Freezing?

Once you have reviewed the information, please call us at 281-443-2362 or email us at [email protected] to set up an appointment.  The receptionist will take your information and our manager Jenny will return your call.  She will then discuss the process and set up the appointment with you.  We are only able to do collect and freezing on certain days so we suggest you schedule far in advance.  Dr. McGuire is our only vet that is able to perform this and she fills up quickly and can only manage at least 1- 2 freezes a day.  The collection part is easy it is the freezing that is very long and tedious.

Scheduled freezes that do not show up will not be allowed to schedule again.  Also, if your dog is unable to be collected or his semen is not viable and cannot be frozen there is still a charge for time and supplies used which is $150.

Источник: http://www.suburbiavet.com/more_info.html

Semen Freezing

Call our team today for more information on our services

Boasting the latest technology in canine breeding and with over 30 years of knowledge and expertise in assisted reprodution, we provide the UK's leading semen collection and and freezing service available to all dog owners across every breed an discipline.

Our semen freezing service has the potential to revolutionise your dog's breeding programme, whilst providing the opportunity for the indefinate preservation of his breeding potential.

Semen Freezing - A Quick Overview

Also known as cryopreservation, semen freezing involves the freezing of semen to a temperature of -196 degrees Celsius. At this temperature, the sperm cells are frozen in a static but viable state and will not degrade or perish providing correct storage and handling is undertaken. Once thawed, the semen is restored to its pre-frozen form, capable of fertilisation and for some dogs, the results can be comparable to that of chilled semen, when frozen, thawed and inseminated correctly.

Semen Freezing – Is It for You? 

Whether you are a dog breeder, or simply a pet owner, semen freezing has many benefits of interest and appeal to every dog owner.
 

1.Preservation of your dog’s breeding potential 

By far one of the key advantages of frozen semen is its indefinite storage, providing for preservation of your dog’s breeding potential, espcially when he is no longer with us. Safeguarding against his unexpected loss or planning for the future, the ability to bank and indefinitely preserve semen should appeal to all dog owners.
 

2.Less stress for your dog and for you

i)For busy showing, competition and working dogs, semen can be taken for freezing during quieter periods, permitting a competition season uninterrupted by breeding duties.

ii)Reduced semen collection frequency – semen freezing provides an alternative/supplement to the demands of natural/chilled semen breeding regimes. With semen freezing the collection frequency can be tailored to the dog's physical needs and semen quality. The bank of semen collected can be used to aid his breeding targets, whether as a complete alternative or supplement to natural/chilled semen collections.

 

3.More efficient use of your dog’s semen

When breeding via natural methods or chilled AI, the dog’s ejaculate is dedicated to one bitch or, in the case of chilled semen, is divided between bitches according to demand with unused semen discarded after 24 hours. With frozen semen, some dogs can split one ejaculate into multiple breeding doses which can be frozen and kept indefinitely, preventing semen wastage and permitting more efficient use of your stud dog. 
 

4.  Logistics and a worldwide market 

i)A further advantage of frozen semen is the ability to ship the semen ahead of the bitch’s planned time of requirement. Most vet practices now have frozen semen storage, enabling bitch owners to order semen weeks, if not months, in advance and so prevent the stress to all parties involved in timing semen arrival against the bitch’s cycle. 

ii)The indefinite shelf life of frozen semen providing its maintenance at -196 degrees Celsius also means it can be shipped worldwide where regulations and transport permits. This opens up the possibility of an exciting worldwide market for you and your dog – the world becomes your dog's oyster with frozen semen distribution.

The Service

Our semen collection and freezing service includes;

  • Semen collection by our experienced team members

  • Processing & freezing into 0.5ml straws in our state of the art laboratory

  • Full post thaw analysis & quality control written report

  • Once the semen is frozen it is transferred into our permanent storage facility, see ‘Frozen Semen Storage & Distribution’ for more details on storage.


How many doses will my dog produce from one collection?

The number of doses that each dog produces is highly variable, the table below shows the estimated doses calculated using body weight.  It may be possible to carry out a supplementary collection on the same day to increase sample size, allowing for greater straw numbers.

 

Small DogUp to 10Kgs1+ Doses 
Medium DogUp to 30Kgs2+ Doses
Large DogUp to 40Kgs3+ Doses
Extra Large Dog40Kgs +4+ Doses

 

This Months Deals!

We run regular semen freezing clinics where you can save over £40 on frozen semen collections. To find out when our next date is click here. 

Have you joined our loyalty scheme yet? click here to claim your FREE semen freeze

 

Please contact us for more information and click here to book.

canine cytology AI K9 dog sperm frozen freeze semen fertility UK Midlands near me kennels insemination clinic breeding services male ovulation progesterone testing reproduction

Источник: https://www.elitekennelfertility.com/serviceoption/19/semen-freezing

Dog sperm bank near me -

Semen Freezing

Call our team today for more information on our services

Boasting the latest technology in canine breeding and with over 30 years of knowledge and expertise in assisted reprodution, we provide the UK's leading semen collection and and freezing service available to all dog owners across every breed an discipline.

Our semen freezing service has the potential to revolutionise your dog's breeding programme, whilst providing the opportunity for the indefinate preservation of his breeding potential.

Semen Freezing - A Quick Overview

Also known as cryopreservation, semen freezing involves the freezing of semen to a temperature of -196 degrees Celsius. At this temperature, the sperm cells are frozen in a static but viable state and will not degrade or perish providing correct storage and handling is undertaken. Once thawed, the semen is restored to its pre-frozen form, capable of fertilisation and for some dogs, the results can be comparable to that of chilled semen, when frozen, thawed and inseminated correctly.

Semen Freezing – Is It for You? 

Whether you are a dog breeder, or simply a pet owner, semen freezing has many benefits of interest and appeal to every dog owner.
 

1.Preservation of your dog’s breeding potential 

By far one of the key advantages of frozen semen is its indefinite storage, providing for preservation of your dog’s breeding potential, espcially when he is no longer with us. Safeguarding against his unexpected loss or planning for the future, the ability to bank and indefinitely preserve semen should appeal to all dog owners.
 

2.Less stress for your dog and for you

i)For busy showing, competition and working dogs, semen can be taken for freezing during quieter periods, permitting a competition season uninterrupted by breeding duties.

ii)Reduced semen collection frequency – semen freezing provides an alternative/supplement to the demands of natural/chilled semen breeding regimes. With semen freezing the collection frequency can be tailored to the dog's physical needs and semen quality. The bank of semen collected can be used to aid his breeding targets, whether as a complete alternative or supplement to natural/chilled semen collections.

 

3.More efficient use of your dog’s semen

When breeding via natural methods or chilled AI, the dog’s ejaculate is dedicated to one bitch or, in the case of chilled semen, is divided between bitches according to demand with unused semen discarded after 24 hours. With frozen semen, some dogs can split one ejaculate into multiple breeding doses which can be frozen and kept indefinitely, preventing semen wastage and permitting more efficient use of your stud dog. 
 

4.  Logistics and a worldwide market 

i)A further advantage of frozen semen is the ability to ship the semen ahead of the bitch’s planned time of requirement. Most vet practices now have frozen semen storage, enabling bitch owners to order semen weeks, if not months, in advance and so prevent the stress to all parties involved in timing semen arrival against the bitch’s cycle. 

ii)The indefinite shelf life of frozen semen providing its maintenance at -196 degrees Celsius also means it can be shipped worldwide where regulations and transport permits. This opens up the possibility of an exciting worldwide market for you and your dog – the world becomes your dog's oyster with frozen semen distribution.

The Service

Our semen collection and freezing service includes;

  • Semen collection by our experienced team members

  • Processing & freezing into 0.5ml straws in our state of the art laboratory

  • Full post thaw analysis & quality control written report

  • Once the semen is frozen it is transferred into our permanent storage facility, see ‘Frozen Semen Storage & Distribution’ for more details on storage.


How many doses will my dog produce from one collection?

The number of doses that each dog produces is highly variable, the table below shows the estimated doses calculated using body weight.  It may be possible to carry out a supplementary collection on the same day to increase sample size, allowing for greater straw numbers.

 

Small DogUp to 10Kgs1+ Doses 
Medium DogUp to 30Kgs2+ Doses
Large DogUp to 40Kgs3+ Doses
Extra Large Dog40Kgs +4+ Doses

 

This Months Deals!

We run regular semen freezing clinics where you can save over £40 on frozen semen collections. To find out when our next date is click here. 

Have you joined our loyalty scheme yet? click here to claim your FREE semen freeze

 

Please contact us for more information and click here to book.

canine cytology AI K9 dog sperm frozen freeze semen fertility UK Midlands near me kennels insemination clinic breeding services male ovulation progesterone testing reproduction

Источник: https://www.elitekennelfertility.com/serviceoption/19/semen-freezing

Collect and Freeze Semen

OUR FREEZING CENTER WILL BE CLOSING AT THE END OF 2021. ALL SEMEN WILL BE SENT TO ZOETIS OR NC STATE VET SCHOOL FOR LONG TERM STORARGE. 

WE ARE CURRENTLY NOT DOING FREEZES, DUE TO THE PANDEMIC. HOWEVER YOU CAN CONTACT NC STATE VET SCHOOL FOR INFORMATION ABOUT THEIR FREEZING CENTER CALL  (919) 513 6300 option 2 or  email to [email protected]

We are a freezing center for Zoetis. We do store semen at our facility. If you are going to be using us in the near future for your dog's reproductive needs, you can go ahead and have semen shipped to us. However please let us know that you are having semen shipped prior to shipping it to us. 


Please contact us for the Collect and Freeze requirements and process. Each collection has to be scheduled in advance, and collections are only done first thing in the mornings. The process takes about 6 hours, even though the collected dog is only in the office for about 30 minutes. 

Collect & Freeze Information: 

Q. What do I need to do to collect semen on my stud dog?

A. That's an easy one! Just call our office at the number listed and we can make you an appointment. Collections for freezing must be done at 9am, no later. It takes 6 hours to take semen thru the freezing process; however the dog is only here for about 20-30 minutes.


Q. What will I need to bring to my appointment?

A. You will need to bring the following:

  1. You are required to bring your AKC or other registration papers verifying that you are a current owner of the stud. (for unregistered dogs this does not apply)
  2. You will need a copy of your DNA Profile, or we can collect the DNA sample and send it in for an additional fee.
  3. You will need a current Brucellosis test.
  4. A semen freezing contract must be completed, since we are a freezing center for Zoetis.

Q. What if I want to send my stud with an agent for collection?

A. Please provide that person with all the items above and payment. Please note that the semen contract MUST be signed by the STUD DOG OWNER. The semen will be collected and stored in Stud owners' name.


Q. Can I let someone else collect my stud and let them own the semen?

A. Yes and No. Semen Ownership can be transferred to persons, not listed as an owner on the registration papers. The semen will need to first be registered to the stud dog owner and then transferred to the new owner.


Q. Is there a cost for annual semen storage?

A. Yes, the annual storage fees are ~$120.00 per year.


Q. How do I arrange to ship my frozen semen?

A. Frozen semen shipping should be planned ahead of time. Please remember that there are a limited number of shipping tanks available for use. It takes 24 hours to charge a shipping dewar. Extra fees do apply for STAT charging and weekend/holiday deliveries.

Источник: https://plantationanimalhospitalnc.com/collect-and-freeze-semen

Independent and combined effects of diethylhexyl phthalate and polychlorinated biphenyl 153 on sperm quality in the human and dog

Abstract

A temporal decline in human and dog sperm quality is thought to reflect a common environmental aetiology. This may reflect direct effects of seminal chemicals on sperm function and quality. Here we report the effects of diethylhexyl phthalate (DEHP) and polychlorinated biphenyl 153 (PCB153) on DNA fragmentation and motility in human and dog sperm. Human and dog semen was collected from registered donors (n = 9) and from stud dogs (n = 11) and incubated with PCB153 and DEHP, independently and combined, at 0x, 2x, 10x and 100x dog testis concentrations. A total of 16 treatments reflected a 4 × 4 factorial experimental design. Although exposure to DEHP and/or PCB153 alone increased DNA fragmentation and decreased motility, the scale of dose-related effects varied with the presence and relative concentrations of each chemical (DEHP.PCB interaction for: DNA fragmentation; human p < 0.001, dog p < 0.001; Motility; human p < 0.001, dog p < 0.05). In both human and dog sperm, progressive motility negatively correlated with DNA fragmentation regardless of chemical presence (Human: P < 0.0001, r = −0.36; dog P < 0.0001, r = −0.29). We conclude that DEHP and PCB153, at known tissue concentrations, induce similar effects on human and dog sperm supporting the contention of the dog as a sentinel species for human exposure.

Introduction

Over the last four decades, there has been increasing concern over declining human male reproductive health. Reduced sperm counts have been widely used as an index of male subfertility and meta-analytical studies indicate a 50% global reduction in quality from 1938 to 20111,2,3. Sperm morphology has also been reported to decrease over a period of 17 years in France with some geographical regional variation; Aquitaine and Midi-Pyrenees having the lowest morphology combined with the lowest concentrations4,5. These data are indicative of an environmental aetiology and, in support of this, epidemiological studies showing increased incidences of testicular cancer and malformations at birth have been linked to regions with reduced sperm counts6,7.

Temporal trends in human semen quality are paralleled by a similar trend in dogs that live in the human household, where sperm motility declined by 30% over a 26 year period8. In this latter study, all data was generated from a single laboratory using consistent techniques and thus did not suffer from changes in methodology and quality assurance over the time span encompassed in human meta-analytical studies9. These observations support the hypothesis that temporal trends in semen quality, both in the human and dog, are due to shared environmental factors and that the dog may be a sentinel for human exposure to such factors. Access to a controlled breeding population of assistance dogs that are routinely sampled for sperm quality provides a cost-effective means of sperm analyses without the stigma and social complications that accompany analogous human studies. Furthermore, there is considerable potential to extend these analyses to any individual or population of dogs. For example, although not investigated in the current study, this could be achieved by semen collection from the tail of the epididymis10 immediately after removal of dog testes at routine surgical neutering; a procedure that is performed on hundreds of thousands of dogs worldwide each year. In addition, semen collections from live dogs is a procedure that is tolerated by a majority of breeds11 unaccustomed to routine fertility monitoring and can therefore easily be carried out by a trained technician.

Declining sperm quality has been linked with the exposure to persistent anthropogenic chemicals, many of which exhibit endocrine disrupting activity6. Although the mechanisms underlying these putative effects are uncertain, historically, the period of fetal development has been highlighted as being particularly sensitive to chemicals with endocrine disrupting activity12. However, a number of studies have shown that environmental chemicals (ECs) are present in semen in a range of species, including the human, raising the possibility of a direct acute effect of chemicals on sperm13,14,15. In support of this theory, an elevated concentration of seminal bisphenol A (BPA) has been associated with infertility in men15,16 and elevated human seminal phthalate metabolites have been associated with reduced sperm counts17. In a separate study, the phthalates DEHP and di-n-butyl-phthalate (DBP) in human semen were reported to be inversely associated with motility and this was confirmed by the direct application of the same phthalates, at seminal concentrations, to sperm in vitro18. By contrast, PCB congeners 118, 126 and 153 were reported to have no negative effect on human sperm motility in vitro, both individually and in combination19. Similar findings have been reported in the dog where DEHP and PCB153, at concentrations detected in dog semen and testis, exhibited inhibitory and stimulatory effects respectively when tested on sperm motility in vitro8. In the same study, both DEHP and PCB153 were detected in a range of dry and wet dog foods indicative of a dietary source. Both DEHP and PCB153 are widely present in the environment and have been detected in tissues/fluids ranging from human breast milk to ovine liver. DEHP is a widely used plasticizer that leaches out into food and liquids and PCBs are lipophilic, and are therefore present in fatty foods20,21,22. Consequently, exposure occurs largely through the diet and these chemicals are deemed as risk factors for reproductive function23,24,25. In support of this, our own published study has shown that DEHP, PCB153 and other PCB congeners are present within both dry and wet dog food sources8 [DEHP: wet and dry food, 0.37 ± 0.10 and 0.20 ± 0.03 μg/g respectively; ∑PCBs: wet and dry food, 1.35 ± 0.5792 and 0.78 ± 0.223 μg/kg respectively; PCB153: wet and dry food, 0.39 ± 0.193 and 0.22 ± 0.11 μg/kg respectively].

Another parameter of ejaculate quality is the proportion of sperm that exhibit DNA fragmentation26. Environmental chemicals have been shown to induce both human and dog sperm DNA fragmentation8,27 and a commercial mixture of PCBs (Arochlor), administered to rats in vivo and added to sperm in vitro, also increased sperm DNA fragmentation28.

In total, these data suggest that environmental chemicals induce similar acute effects on human and dog sperm in vitro: measurement of sperm motility and DNA fragmentation are tried and tested measures of such chemical effects. Since sperm concentration would not alter during the period of in vitro culture and morphology is confounded by abnormality classification, partly due to swelling that may occur during culture and the generation of artefacts during processing, these parameters were not selected for testing acute chemical effects in vitro29,30. Notwithstanding, testing the effects of individual chemicals on sperm functional parameters does not represent “real-life” exposure to a mixture of chemicals, many of which exhibit synergistic, antagonistic or additive effects. Two chemicals known to be present in dog seminal plasma and testis were therefore selected and their effects tested both independently and in combination, on sperm motility and DNA fragmentation in both the human and dog.

Results

Chemical effects on percentage normal sperm motility in human and dog

The analyses of both the human and the dog sperm motility data found the interaction terms between PCB and DEHP to be significant (human p < 0.001; dog p < 0.05). This indicates that the dose response to either chemical was not independent of the level of the other chemical present. Figure 1 illustrates the effects of PCB153 and DEHP, individually and combined, on dog and human sperm motility. In dog sperm, PCB153 in the absence of DEHP induced a dose-dependent inhibitory effect on sperm motility (Fig. 1ai). In the presence of DEHP at 2x and 10x mean testis concentration (MTC), no dose dependent inhibitory effect of PCB153 was observed. However, reduced motility was observed in the presence of 100x DEHP co-incubated with 2x and 10x PCB153 (Fig. 1aii).

Effect of DEHP and PCB153 on dog and human sperm motility. Chemicals tested individually [ai,bi: PCB only; aii,bii: grey bars: DEHP only] and in combination [aii,bii]. Graphs display fixed concentrations of DEHP with increasing concentrations of PCB153 in dog [ai,aii: p <  0.01] and human [bi,bii: p <  0.001] sperm. Grade a motility: >25 μm/s Error bar = 1 Standard Error of Difference. MTC = Mean testis concentration.

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In contrast to the dog, neither PCB153 nor DEHP, in the absence of the other chemical, influenced human sperm motility (Fig. 1bi,bii). However, in the presence of 2x DEHP, a dose dependent inhibitory effect in response to PCB153 was observed (Fig. 1bii). The inhibitory effect of PCB153 was broadly maintained in the presence of 10x and 100x DEHP with the exception of 10x PCB153/10x DEHP and 100x PCB153/100x DEHP, where no inhibition was observed (Fig. 1bii).

Chemical effects on sperm DNA integrity in human and dog

For both the human and dog DNA fragmentation data, significant (P < 0.001) PCB.DEHP interaction terms were found. Again, this indicates that the response of DNA integrity to one chemical is not independent of the presence or level of the other chemical. Figure 2 illustrates the effects of PCB153 and DEHP, individually and combined, on both dog and human sperm DNA fragmentation using the sperm chromatin dispersion assay. In the dog, PCB153 in the absence of DEHP induced a dose-dependent increase in sperm DNA fragmentation (Fig. 2ai). A similar dose-dependent increase in DNA fragmentation was observed in response to DEHP in the absence of PCB153 (Fig. 2aii). When PCB153 and DEHP were tested in combination, DNA fragmentation was still increased at higher concentrations of PCB153 but the dose-dependent response was blunted (Fig. 2aii). The response of human sperm to PCB153 and DEHP independently and combined, paralleled that observed in the dog. Figure 2b illustrates that human sperm incubated with PCB153 or DEHP in the absence of the other chemical, exhibited a dose-dependent increase in DNA fragmentation (Fig. 2bi,bii). In addition, DNA fragmentation was increased in response to both chemicals tested in combination although additive effects were not apparent.

Effect of DEHP and PCB153 on dog and human sperm DNA fragmentation. Chemicals tested individually [ai,bi: PCB only; aii,bii: grey bars: DEHP only] and in combination [aii,bii]. Graphs display fixed concentrations of DEHP with increasing concentrations of PCB153 in dog [ai,aii: p ≤ 0.001] and human [bi,bii: p ≤ 0.001] sperm. Error bar = 1 Standard Error of difference between means. MTC = Mean testis concentration.

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Sperm DNA fragmentation and motility correlations

Figure 3 illustrates the relationship between progressive motility and DNA fragmentation in the presence and absence of each chemical independently and combined. Despite chemical effects on sperm motility (Fig. 1) and DNA fragmentation (Fig. 2), the relationship between these two parameters remained the same regardless of the nature of the chemical exposure. All correlations were significant except the human control samples [Dog (Fig. 3i, n = 352): Control; p < 0.05, r = −0.527, n = 22; DEHP; p < 0.05, r = −0.2862, n = 66; PCB-153; p < 0.01, r = −0.3276, n = 66; Mixture; p < 0.0001, r = −0.2826, n = 198; vs Human (Fig. 3ii, n = 288): Control; p > 0.05, r = −0.4374, n = 18; DEHP; p < 0.05, r = −0.3118, n = 54; PCB-153; p < 0.05, r = −0.2994, n = 54; Mixture; p < 0.0001, r = −0.4037, n = 162].

Correlation between progressive motility and DNA fragmentation in both dog and human sperm. Values from 32 sperm assessments (16 treatments, two time points). Each point represents a different sperm culture equating to a total n = 352 [dog] and n = 288 [human]. Colours denote culture media constituents to demonstrate spread: Control (black), DEHP (red), PCB153 (blue) and mixture (green). Dog (i, n = 352): Control; p < 0.05, r = −0.527, n = 22; DEHP; p < 0.05, r = −0.2862, n = 66; PCB153; p < 0.01, r = −0.3276, n = 66; Mixture; p < 0.0001, r = −0.2826, n = 198; vs Human (ii, n = 288): Control; p > 0.05, r = −0.4374, n = 18; DEHP; p < 0.05, r = −0.3118, n = 54; PCB153; p < 0.05, r = −0.2994, n = 54; Mixture; p < 0.0001, r = −0.4037, n = 162]. Confidence bands (95%) plotted for visual purposes only. Progressive motility based on WHO pre-2010 where sperm swimming grades a and b are combined: ≥5 µm/s.

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Discussion

Data presented in this paper are significant because we conclusively demonstrate that a selected phthalate (DEHP) and PCB congener (PCB153), at concentrations relevant to environmental exposure, reduce dog sperm motility and increase DNA fragmentation in vitro. In the human, our data showing reduced motility and increased DNA fragmentation are indicative of similar sensitivities to these chemicals in both species. Furthermore, when the chemicals were combined and co-incubated with dog or human sperm, the overall impact on motility or DNA fragmentation was dependent on the concentration ratio. This is the first study to select two environmental chemicals and to test them at four concentrations in all possible combinations representative of those found in the male reproductive tract and fluids. In addition, a negative correlation between motility and DNA fragmentation, that incorporates chemical variables, is described in both species.

Our data on the sensitivity of sperm to short term chemical exposure support similar studies using a range of environmentally relevant chemical challenges in vitro. For example, DEHP, DBP and mono-n-butyl phthalate (MBP), are reported to reduce sperm motility in vitro when added at seminal concentrations measured in infertile men18,31. A mixture of PCBs are also reported to reduce sperm motility in both the human and pig32,33 and in the human, p,p’-dichloro-diphenyl-dichloro-ethylene (p,p’-DDE) has been shown to increase Ca2+ uptake by sperm in a mechanism that involves the CatSper channel34.

To minimise the effects of pre-exposure to chemicals present in ejaculates collected for culture, the current study used a concentration range that encompassed the reported variability in seminal concentrations and reduced pre-existing chemical concentrations by sperm processing and washing prior to culture. The subsequent comparison to controls, with no further chemical added, provided a means of testing chemical effects. Despite these steps, pre-exposure differences will inevitably exist between, and within, species. For example, in the current study, when DEHP is present at twice the concentration found in testis, dog sperm appears to be less sensitive to lower PCB concentrations than human sperm. In populations of men from Greenland, Sweden, Poland and the Ukraine, increased seminal PCB153 has been consistently associated with reduced sperm motility but not concentration or morphology35,36. In addition, sperm DNA fragmentation was positively associated with PCB153 in European populations but not in samples from Greenlandic men: an observation that likely reflects different pre-exposures.

In the current study, our intention was not to measure and equate chemical concentrations in each individual sample with sperm motility and fragmentation, but to use a concentration range previously established in the dog as a ballpark estimate of chemical concentrations in the male reproductive tract8. That is, concentrations found in the dog reproductive tract and seminal plasma, used as an indicator of those likely present in the human. In support of this contention, human seminal PCB (total) and DEHP concentrations reported in populations of fertile men [total PCB: up to 5.8 ng/ml, DEHP: mean of 0.61 μg/ml]37,38 are comparable to those detected in the dog [PCB: 0.26–13.2 ng/ml; DEHP: 0.75–37.5 μg/ml]8.

Notably, elevated concentrations of both chemicals have been linked with reduced human male fertility18. Semen PCB concentrations have been reported to be higher in a population of ‘infertile’ men (parameters stated as <20 million/ml or <25% progressive motility and/or <30% normal morphology)25. Further, a research group in China reported an association between increased urinary phthalate metabolites, reduced sperm count and increased sperm DNA damage14. Although the mechanism was not proven, the authors suggest that this likely reflects chemical effects on testicular Sertoli and germ cells as reported in animal studies39,40,41,42.

Although both DEHP and PCB153 have been reported to reduce sperm motility and increase sperm DNA fragmentation individually in the human8,18,37, they have not been assessed in combination, at environmentally relevant concentrations, as reported here. A limited number of studies have investigated some combinations of environmental chemicals for their effect on human sperm function32. Real-life exposure is to a complex mixture of chemicals that are likely to exhibit synergistic or antagonistic, as well as additive effects, on sperm. Mixtures of endocrine disrupting chemicals have been shown to cooperatively increase Ca2+ concentrations in sperm through the activation of the principle calcium channel CatSper43.

In the current study, the mechanisms that underlie the concentration ratio-dependent effects of the two chemicals combined remain uncertain. PCBs and DEHP are considered to be pro-estrogenic and anti-androgenic respectively raising the possibility that a change in concentration ratio may alter the relative activation of sperm estrogen or androgen receptors44,45. Indeed, in a population of ‘infertile’ men (parameters stated as sperm count < 20 × 106, motility < 50%, morphology < 14% normal) androgen receptor expression is reported to positively correlate with sperm motility46 and PCBs are reported to affect sperm concentration and motility relative to the number of CAG repeats in the androgen receptor gene47. This may account for PCB influences on motility even in the presence of DEHP. Estrogenic compounds have also been reported to reduce human sperm motility in vitro via an induction in redox activity and this mechanism has also been linked to the induction of DNA fragmentation by 2-hydroxy estradiol48.

The greater consistency of chemical effects on human and dog sperm DNA fragmentation compared to motility is interesting and emphasises the importance of looking at more than one sperm functional/viability parameter when assessing environmental effects. It is important to note however that despite this subtle difference, the two sperm parameters were highly correlated in both species and remained so in the presence of the chemicals independently and combined. This is an important observation since it has been reported that human male infertility is associated with increased levels of sperm DNA damage and that sperm motility defects are highest in samples with increased DNA fragmentation49,50. This raises the possibility that there may be a similar relationship between sperm DNA fragmentation, motility and fertility in the dog.

In conclusion, we have demonstrated that the use of low dose tissue relevant concentrations of DEHP and PCB153, independently and in combination, negatively impact on sperm motility and DNA fragmentation in samples obtained from humans and dogs. Since these effects are broadly similar in both species, this raises the possibility that the environmental impact of chemicals in the dog may provide a means of investigating pollutant effects on mammalian fertility in a species in which external influences, such as diet, are better controlled than in an equivalent human study.

Methods

Ethical Approval

Human

Semen donations were obtained from anonymous HFEA registered donors (n = 9) attending the fertility unit at Nottingham University Hospitals. Donors provided informed consent for the use of samples in this research project ensuring ‘General Data Protection Regulation’ compliance. Each donor was initially screened following HFEA and British Fertility Society protocols51. No samples from fertility patients were used and all donors completed HFEA consent forms. Ethical approval was obtained from the School of Veterinary Medicine Ethical Review Committee [Reference 1511,150723]. The HFEA consent forms completed by donors and ethical approval documents were also approved by the Chair of the Ethical Review Committee of the Faculty of Medicine and Health Sciences, University of Nottingham. In accordance with the Royal College of Pathologists guidance on the use of pathological specimens [https://www.rcpath.org/resourceLibrary/the-retention-and-storage-of-pathological-records-and-specimens-5th-edition-.html], no further ethical approval was required.

Dog

Semen was collected as part of routine reproductive examination of stud dogs subject to owner consent with full GDPR compliance. Due to dog sperm being collected as part of routine reproductive health checks, the economical outlay associated with sample collection was minimised. The dogs resided in the same region of the UK and lived in the modern household with owners briefed on use of controlled diet and exercise regimes. All semen collections were performed in accordance with relevant guidelines and regulations and the collection protocol was approved by the School of Veterinary Medicine Ethical Review Committee (Refs: 208 101012, 513 120117 and 1097 140227).

Human sperm collection and preparation

Samples were liquefied for 20 minutes at room temperature prior to semen preparation. Ejaculate underwent density gradient centrifugation using a Universal 320 R Hettich centrifuge (DJB Labcare, Newport Pagnell, UK) at 25 °C. Briefly, one millilitre of 40% isotonic density gradient was loaded onto one millilitre of 80% isotonic medium [PureSperm 40/80, Nidacon, Sweden]. Two millilitres of liquefied semen were loaded onto both isotonic mediums and reagents centrifuged at 300 × g for 22 minutes. On completion, the sperm rich pellet was re-suspended using 1.5 ml PureSperm wash (pH range of 7.3–8.5; osmolality: 290–300 mOsm/kg H2O, Nidacon, Sweden).

Dog sperm collection

Ejaculate was collected from stud dogs (n = 11) by routine digital manipulation. The sperm rich fraction (fraction 2) was collected into sterile plastic 15 millilitre Greiner centrifuge tubes [Sigma-Aldrich, Dorset, UK]. INRA extending medium [INRA, Nouzilly, France] was added to sperm at a 2:1 ratio to aid sperm survival.

Chemical preparation

The two chemicals selected for co-culture with sperm were diethylhexyl phthalate (DEHP) and polychlorinated biphenyl congener 153 (PCB153) and concentrations were calculated relative to those present in dog testicular tissue8. The rationale for this was (1) dog testis concentrations of both chemicals have been established and standardised in our previous study and are reflective of exposure of the male reproductive tract8 (2) dog and human semen PCB concentrations are variable but generally higher than testis and in range of the concentrations tested in vitro8,25,52 (3) although dog semen DEHP concentrations have not been determined, due to the large amount of dry material required, reported human DEHP concentrations are in range of established dog testis measurements14,18,53. On this basis, dog testis concentrations were used as a standardised measure of environmental exposure relevant to both species. DEHP (CAS no: 117-81-7) and PCB153 (CAS no: 35065-27-1) [Sigma-Aldrich, Dorset, U.K.] were dissolved in 100% dimethylsulphoxide (DMSO) and diluted with PBS to 4x, 20x and 200x mean testis concentration containing 0.02% DMSO. Chemical preparations were co-incubated with sperm at a 1:1 ratio with a final exposure concentration of 2x, 10x and 100x mean testis concentration. Sperm were incubated with each chemical at each concentration individually and with a mixture of the two chemicals in all 16 combination ratios (Table 1). Acute treatment effects were assessed at 10 minutes and 3 hours and control incubations carried out with 0.01% DMSO only.

Full size table

Motility assessment

Sperm motility was assessed using Computer Assisted Sperm Analysis (CASA) software. Sperm were acclimatised for two minutes on a 37 °C stage prior to assessment. Five µl of sperm, irrespective of species, were pipetted into a specialised 20 µl cellvision glass slide counting chamber (Code CV1020-2CV; CellVision, the Netherlands). A minimum of 200 sperm were assessed for each treatment. For human sperm, motility was tracked by use of the diagnostic software ‘SAMi’ (Procreative Diagnostics Ltd, Staffordshire, UK) and an Olympus BH2 Microscope [KeyMed (Medical and Industrial Equipment) Ltd, Essex, UK]. Dog sperm motility was assessed using the Hobson’s CASA tracking system (Hobson’s tracking systems Ltd., Sheffield, UK) and viewed using a negative-high phase contrast objective (x20) on an Olympus BH2 microscope fitted with a camera ocular. For both species, sperm motility was assessed according to WHO 199954 where grade a motility was classified as ≥ 25 µm/s and progressive motility ≥ 5 µm/s (grades a & b combined).

DNA fragmentation

Sperm DNA fragmentation was quantified by defragmentation index (sDFI %) measured using the sperm chromatin dispersion assay55. Sperm were assessed based on the size of the halos, indicative of sperm that exhibited nuclei with non-fragmented DNA. Sperm with denatured or fragmented DNA were identified by the absence of a halo, or a halo that was exceedingly small. Briefly, equal volumes of intact, unfixed sperm and 1% agarose solution were combined. Fifteen microliter aliquots of sperm-agarose suspensions were pipetted onto agarose pre-coated poly-L-lysine slides (CAS: P4981; ThermoFisher Scientific Ltd. UK), covered with a 22 mm × 22 mm cover-slip, and placed in a 4 °C environment for five minutes to fix the sample. After an acid treatment (0.08 M HCl) of seven minutes, sperm were incubated in a lysing reagent (0.8 M DTT, 0.4 M Tris, 2 M NaCl, 1% triton-X) for 20 minutes. Slides were then submerged in dH2O for a period of five minutes followed by dehydration through a series of ethanol solutions (70%, 90% and 100% respectively). Visualisation was obtained using Diff-Quick staining reagents (eosinophilic and basophilic stains; CAS 9990700, ThermoFisher Scientific Ltd, Paisley, UK) and analysis undertaken using oil immersion, at 1000x magnification [Leica DM 5000 B microscope; Leica Microsystems, Milton Keynes, UK]. A minimum of 200 sperm were assessed for each treatment. Positive controls were incubated with 300 µm H2O2 before denaturation and negative controls by omission of the denaturation step. To determine the defragmentation index, the number of fragmented sperm was divided by the total number of sperm counted. This provided a value for the proportion of sperm that were fragmented. This proportion was then plotted on the logit scale.

Experimental design and Statistical analysis

For both the human and dog experiments, sperm samples were treated with PCB153 at one of four concentrations in combination with DEHP, also at one of four concentrations. The concentrations used for both chemicals corresponded to 0x, 2x, 10x and 100x the baseline concentration measured from dog testes. Thus there were 16 possible combinations of PCB153 and DEHP. Each combination was randomly allocated to one of 16 separate sub-samples of sperm, from each replicate donor.

Statistical analysis was undertaken using GenStat 17th edition (VSN International Ltd, Hempstead, UK). The proportions out of known numbers of sperm that had a particular characteristic were analysed as grouped binary data by fitting a generalised linear mixed model with a logit link function, assuming a binomial error distribution. The fixed effects included in the statistical model were PCB, DEHP and the PCB.DEHP interaction term. Another fixed effect, TIME, and its interaction terms were included when two repeated samples from the same tube were measured three hours apart. The random effects were donor and culture-tube within donor.

The statistical significance of a fixed effect was tested using an F-Ratio test. The analysis output provided predicted mean proportions and standard errors of difference between means which were graphically represented on a logit scale. Logit values were converted back into proportions and graphically plotted [GraphPad Prism 7.0, GraphPad Ltd, California, USA]. A single error bar on each figure represents the standard error of the difference between means. Where correlations were investigated, data was assumed to be non-normally distributed and a Spearman’s rank correlation analysis was undertaken to provide correlation coefficients between motility and DNA fragmentation.

Data Availability

The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

References

  1. 1.

    Carlsen, E., Giwercman, A., Keiding, N. & Skakkebaek, N. E. Evidence for decreasing quality of semen during past 50 years. BMJ305, 609–613 (1992).

    CASArticle Google Scholar

  2. 2.

    Swan, S. H., Elkin, E. P. & Fenster, L. The question of declining sperm density revisited: an analysis of 101 studies published 1934–1996. Environ. Health Perspect.108, 961–966 (2000).

    CASArticle Google Scholar

  3. 3.

    Levine, H. et al. Temporal trends in sperm count: a systematic review and meta-regression analysis. Hum. Reprod. Update23, 646–659 (2017).

    Article Google Scholar

  4. 4.

    Rolland, M., Le Moal, J., Wagner, V., Royere, D. & De Mouzon, J. Decline in semen concentration and morphology in a sample of 26,609 men close to general population between 1989 and 2005 in France. Hum. Reprod. 28, 462–470 (2013).

  5. 5.

    Le Moal, J. et al. Semen quality trends in French regions are consistent with a global change in environmental exposure. Reproduction147, 567–574 (2014).

    Article Google Scholar

  6. 6.

    Bay, K., Asklund, C., Skakkebaek, N. E. & Andersson, A.-M. Testicular dysgenesis syndrome: possible role of endocrine disrupters. Best Pract. Res. Clin. Endocrinol. Metab.20, 77–90 (2006).

    CASArticle Google Scholar

  7. 7.

    Skakkebaek, N. E. Sperm counts, testicular cancers, and the environment. BMJ (Clinical research ed.)359, j4517 (2017).

    Article Google Scholar

  8. 8.

    Lea, R. G. et al. Environmental chemicals impact dog semen quality in vitro and may be associated with a temporal decline in sperm motility and increased cryptorchidism. Sci. Rep.6, 31281 (2016).

    ADSCASArticle Google Scholar

  9. 9.

    Pacey, A. A. Are sperm counts declining? Or did we just change our spectacles? Asian J. Androl.15, 187–190 (2013).

    Article Google Scholar

  10. 10.

    Ponglowhapan, S., Chatdarong, K., Sirivaidyapong, S. & Lohachit, C. Freezing of epididymal spermatozoa from dogs after cool storage for 2 or 4 days. Theriogenology66, 1633–1636 (2006).

    CASArticle Google Scholar

  11. 11.

    Hermansson, U. & Linde Forsberg, C. Freezing of stored, chilled dog spermatozoa. Theriogenology65, 584–593 (2006).

    Article Google Scholar

  12. 12.

    Lea, R. G. et al. Endocrine disruptors and ovine reproductive development. In Reproduction in Domestic Ruminants Vol.VIII (eds Juengel, J. et al.) 209–227 (Context Products Ltd., UK, 2014).

  13. 13.

    Kamarianos, A., Karamanlis, X., Theodosiadou, E., Goulas, P. & Smokovitis, A. The presence of environmental pollutants in the semen of farm animals (bull, ram, goat, and boar). Reprod. Toxicol.17, 439–445 (2003).

    CASArticle Google Scholar

  14. 14.

    Wang, Y.-X. et al. Phthalate exposure and human semen quality: Results from an infertility clinic in China. Environ. Res.142, 1–9 (2015).

    ADSCASArticle Google Scholar

  15. 15.

    La Rocca, C. et al. Exposure to Endocrine Disruptors and Nuclear Receptors Gene Expression in Infertile and Fertile Men from Italian Areas with Different Environmental Features. Int. J. Environ. Res. Public Health12, 12426–12445 (2015).

    Article Google Scholar

  16. 16.

    Vitku, J. et al. Difflerences in bisphenol A and estrogen levels in the plasma and seminal plasma of men with different degrees of infertility. Physiol. Res.64(Suppl 2), S303–11 (2015).

    CASPubMed Google Scholar

  17. 17.

    Chang, W.-H., Wu, M.-H., Pan, H.-A., Guo, P.-L. & Lee, C.-C. Semen quality and insulin-like factor 3: Associations with urinary and seminal levels of phthalate metabolites in adult males. Chemosphere173, 594–602 (2017).

    ADSCASArticle Google Scholar

  18. 18.

    Pant, N. et al. Environmental and experimental exposure of phthalate esters: the toxicological consequence on human sperm. Hum. Exp. Toxicol.30, 507–514 (2011).

    CASArticle Google Scholar

  19. 19.

    Pflieger-Bruss, S. et al. Effects of single non-ortho, mono-ortho, and di-ortho chlorinated biphenyls on human sperm functions in vitro. Reprod. Toxicol.21, 280–284 (2006).

    CASArticle Google Scholar

  20. 20.

    Kastner, J., Cooper, D. G., Maric, M., Dodd, P. & Yargeau, V. Aqueous leaching of di-2-ethylhexyl phthalate and ‘green’ plasticizers from poly(vinyl chloride). Sci. Total Environ.432, 357–364 (2012).

    ADSCASArticle Google Scholar

  21. 21.

    Erythropel, H. C., Maric, M., Nicell, J. A., Leask, R. L. & Yargeau, V. Leaching of the plasticizer di(2-ethylhexyl)phthalate (DEHP) from plastic containers and the question of human exposure. Appl. Microbiol. Biotechnol.98, 9967–9981 (2014).

    CASArticle Google Scholar

  22. 22.

    Faroon, O. & Ruiz, P. Polychlorinated biphenyls: New evidence from the last decade. Toxicol. Ind. Health32, 1825–1847 (2016).

    CASArticle Google Scholar

  23. 23.

    Pocar, P. et al. Exposure to di(2-ethyl-hexyl) phthalate (DEHP) in utero and during lactation causes long-term pituitary-gonadal axis disruption in male and female mouse offspring. Endocrinology153, 937–948 (2012).

    CASArticle Google Scholar

  24. 24.

    Oskam, I. C. et al. Effects of long-term maternal exposure to low doses of PCB126 and PCB153 on the reproductive system and related hormones of young male goats. Reproduction130, 731–742 (2005).

    CASArticle Google Scholar

  25. 25.

    Rozati, R., Reddy, P. P., Reddanna, P. & Mujtaba, R. Role of environmental estrogens in the deterioration of male factor fertility. Fertil. Steril.78, 1187–1194 (2002).

    Article Google Scholar

  26. 26.

    Rex, A. S., Aagaard, J. & Fedder, J. DNA fragmentation in spermatozoa: a historical review. Andrology5, 622–630 (2017).

    CASArticle Google Scholar

  27. 27.

    Evenson, D. P. & Wixon, R. Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA). Toxicol. Appl. Pharmacol.207, 532–537 (2005).

    Article Google Scholar

  28. 28.

    Aly, H. A. A. Aroclor 1254 induced oxidative stress and mitochondria mediated apoptosis in adult rat sperm in vitro. Environ. Toxicol. Pharmacol.36, 274–283 (2013).

    CASArticle Google Scholar

  29. 29.

    Mortimer, D. & Menkveld, R. Sperm morphology assessment—historical perspectives and current opinions. J. Androl.22, 192–205 (2001).

    CASPubMed Google Scholar

  30. 30.

    Boersma, A., Rasshofer, R. & Stolla, R. Influence of sample preparation, staining procedure and analysis conditions on bull sperm head morphometry using the morphology analyser integrated visual optical system. Reprod. Domest. Anim.36, 222–229 (2001).

    CASArticle Google Scholar

  31. 31.

    Xie, F. et al. Effects of two environmental endocrine disruptors di-n-butyl phthalate (DBP) and mono-n-butyl phthalate (MBP) on human sperm functions in vitro. Reprod. Toxicol.83, 1–7 (2019).

    CASArticle Google Scholar

  32. 32.

    Jiang, L.-G. et al. Toxic effects of polychlorinated biphenyls (Aroclor 1254) on human sperm motility. Asian J. Androl.19, 561–566 (2017).

    CASArticle Google Scholar

  33. 33.

    Campagna, C., Guillemette, C., Ayotte, P. & Bailey, J. L. Effects of an environmentally relevant organochlorine mixture and a metabolized extract of this mixture on porcine sperm parameters in vitro. J. Androl.30, 317–324 (2009).

    CASArticle Google Scholar

  34. 34.

    Tavares, R. S., Escada-Rebelo, S., Correia, M., Mota, P. C. & Ramalho-Santos, J. The non-genomic effects of endocrine-disrupting chemicals on mammalian sperm. Reproduction151, R1–R13 (2016).

    CASArticle Google Scholar

  35. 35.

    Toft, G. Persistent organochlorine pollutants and human reproductive health. Dan. Med. J.61, B4967 (2014).

    PubMed Google Scholar

  36. 36.

    Toft, G. et al. Semen quality and exposure to persistent organochlorine pollutants. Epidemiology17, 450–458 (2006).

    Article Google Scholar

  37. 37.

    Bush, B., Bennett, A. H. & Snow, J. T. Polychlorobiphenyl congeners, p,p’-DDE, and sperm function in humans. Arch. Environ. Contam. Toxicol.15, 333–341 (1986).

    CASArticle Google Scholar

  38. 38.

    Han, S. W. et al. An exposure assessment of di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) in human semen. J. Toxicol. Environ. Heal. Part A72, 1463–1469 (2009).

    CASArticle Google Scholar

  39. 39.

    Jones, S., Boisvert, A., Francois, S., Zhang, L. & Culty, M. In utero exposure to di-(2-ethylhexyl) phthalate induces testicular effects in neonatal rats that are antagonized by genistein cotreatment. Biol. Reprod.93(92), 1–14 (2015).

    CAS Google Scholar

  40. 40.

    Richburg, J. H. & Boekelheide, K. Mono-(2-ethylhexyl) phthalate rapidly alters both Sertoli cell vimentin filaments and germ cell apoptosis in young rat testes. Toxicol. Appl. Pharmacol.137, 42–50 (1996).

    CASArticle Google Scholar

  41. 41.

    Ungewitter, E. et al. From the Cover: Teratogenic Effects of in Utero Exposure to Di-(2-Ethylhexyl)-Phthalate (DEHP) in B6:129S4 Mice. Toxicol. Sci.157, 8–19 (2017).

    CASPubMedPubMed Central Google Scholar

  42. 42.

    Zhang, T. et al. Melatonin protects prepuberal testis from deleterious effects of bisphenol A or diethylhexyl phthalate by preserving H3K9 methylation. J. Pineal Res.65, e12497 (2018).

    Article Google Scholar

  43. 43.

    Schiffer, C. et al. Direct action of endocrine disrupting chemicals on human sperm. EMBO Rep.15, 758–765 (2014).

    CASArticle Google Scholar

  44. 44.

    Aquila, S. et al. Estrogen receptor (ER)alpha and ER beta are both expressed in human ejaculated spermatozoa: evidence of their direct interaction with phosphatidylinositol-3-OH kinase/Akt pathway. J. Clin. Endocrinol. Metab.89, 1443–1451 (2004).

    CASArticle Google Scholar

  45. 45.

    Aquila, S. et al. Human sperm express a functional androgen receptor: effects on PI3K/AKT pathway. Hum. Reprod.22, 2594–2605 (2007).

    CASArticle Google Scholar

  46. 46.

    Zalata, A. A. et al. Androgen receptor expression relationship with semen variables in infertile men with varicocele. J. Urol.189, 2243–2247 (2013).

    CASArticle Google Scholar

  47. 47.

    Giwercman, A., Rylander, L. & Lundberg Giwercman, Y. Influence of endocrine disruptors on human male fertility. Reprod. Biomed. Online15, 633–642 (2007).

    CASArticle Google Scholar

  48. 48.

    Bennetts, L. E. et al. Impact of estrogenic compounds on DNA integrity in human spermatozoa: evidence for cross-linking and redox cycling activities. Mutat. Res.641, 1–11 (2008).

    ADSCASArticle Google Scholar

  49. 49.

    Velez de la Calle, J. F. et al. Sperm deoxyribonucleic acid fragmentation as assessed by the sperm chromatin dispersion test in assisted reproductive technology programs: results of a large prospective multicenter study. Fertil. Steril.90, 1792–1799 (2008).

    Article Google Scholar

  50. 50.

    Belloc, S. et al. Sperm deoxyribonucleic acid damage in normozoospermic men is related to age and sperm progressive motility. Fertil. Steril.101, 1588–1593 (2014).

    CASArticle Google Scholar

  51. 51.

    Hamilton, M. et al. Working Party on Sperm Donation Services in the UK. Hum. Fertil.11, 147–158 (2008).

    Article Google Scholar

  52. 52.

    Dallinga, J. W. et al. Decreased human semen quality and organochlorine compounds in blood. Hum. Reprod.17, 1973–1979 (2002).

    CASArticle Google Scholar

  53. 53.

    Zhang, Y.-H., Zheng, L.-X. & Chen, B.-H. Phthalate exposure and human semen quality in Shanghai: a cross-sectional study. Biomed. Environ. Sci.19, 205–209 (2006).

    CASPubMed Google Scholar

  54. 54.

    WHO. Laboratory Manual for the examination of human semen and sperm – cervical mucus interaction. (Cambridge University Press, Cambridge, 1999).

  55. 55.

    Fernandez, J. L. et al. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil. Steril.84, 833–842 (2005).

    CASArticle Google Scholar

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Acknowledgements

We thank Karen Pooley for the management of the human donors attending the Fertility Unit at Nottingham University Hospital and for expert technical advice and assistance. We also thank Natasha White for the collection of ejaculates from a population of assistance dogs. The work was supported by University of Nottingham internal funds.

Author information

Author notes
  1. Rebecca N. Sumner

    Present address: Hartpury University, Gloucester, GL19 3BE, UK

Affiliations

  1. School of Veterinary Medicine & Science, University of Nottingham, Sutton Bonington, LE12 5RD, UK

    Rebecca N. Sumner, Gary C. W. England & Richard G. Lea

  2. Fertility Unit, East Block B floor, Nottingham University Hospital, Nottingham, NG7 2UH, UK

    Mathew Tomlinson

  3. School of Biosciences, University of Nottingham, Sutton Bonington, LE12 5RD, UK

    Jim Craigon

Contributions

R.G.L. and R.N.S. conceived of and designed the study. M.T. managed the human donors and collection of human semen samples and G.C.W.E. managed the collection of canine samples. All authors provided academic input into the paper. R.N.S. carried out the computer assisted sperm analysis, chromatin dispersion assay and subsequent analysis. J.C. provided expert statistical advice and contributed to the analysis. R.G.L. and R.N.S. wrote the paper.

Corresponding author

Correspondence to Richard G. Lea.

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Sumner, R.N., Tomlinson, M., Craigon, J. et al. Independent and combined effects of diethylhexyl phthalate and polychlorinated biphenyl 153 on sperm quality in the human and dog. Sci Rep9, 3409 (2019). https://doi.org/10.1038/s41598-019-39913-9

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Call Us: (+1) 508-875-7086
Slade Veterinary HospitalSlade Veterinary Hospital

Male Breeding Soundness Exam

We offer comprehensive breeding soundness examinations for your stud dogs. This includes a complete physical examination with special attention to the testicles, penis, and prostate, as well as a complete semen evaluation. We review your male's prior breeding history, including litters produced and bitches that missed. Additional testing may include baseline labwork, prostatic and testicular ultrasounds, cultures, and genetic testing.

For questions related to breeding soundness exams, please speak to one of our reproduction technicians – Linn, Kirsten, or Scott.

Semen Collection and Evaluation

The manual collection of semen is a vital component of evaluating your stud dog. The libido of your dog and the quality of the semen sample is improved by the presence of a teaser bitch. You may bring your own teaser or we can provide one for him. We separately collect the different fractions of the ejaculate and utilize a computer-assisted sperm analysis (CASA) machine for thorough evaluation. Our phase contrast microscope and CASA software is state-of-the-art and incredibly precise, providing you with the most detailed information. You will receive a detailed report of your stud dog’s semen analysis.

Your male's semen quality should be evaluated in the month prior to an expected breeding. If he is used frequently, he should be manually collected and evaluated every six months, or any time a breeding does not take.

Options for Semen

Fresh semen

We collect your male, add semen extenders to enhance the motility and lifespan of the ejaculate, and use the sample for breeding immediately with a bitch.

Fresh-chilled semen

This method of collecting semen, adding extender, slowly cooling, and packaging the specimen for shipment has made long distance breeding possible. The stud dog is manually collected at our office when the owner of the bitch notifies you that their female has reached her fertile period. With fresh-chilled semen, it is the semen, not the bitch, that does the traveling. We can arrange same-day or overnight shipment within the continental United States Monday through Saturday. We recommend that stud dog owners have their dog's semen checked for its ability to withstand the chilling process. Please see semen shipping (below) for more information.

Frozen

The purpose of freezing your dog's semen is to ensure his breeding availability. Frozen semen is used when a dog is no longer fertile, deceased, or unavailable due to a scheduling conflict. Please see semen cryopreservation (freezing) below for more information.

Semen Cryopreservation (Freezing)

Our hospital began working with International Canine Genetics in the early 1990s. We are a licensed freezing center and offer semen freezing once a month. Special arrangements can be made to mitigate scheduling conflicts. 

Consider freezing your dog's semen to:

  • Preserve his genetics for your own future breeding program
  • Provide stud service when your dog is not available (show/trial schedules, injury, illness)
  • Use as back-up for breeding involving winter shipping or difficult travel
  • Use for international breeding (Please contact us in advance for individual export regulations.)

The ideal time to collect your dog's semen for freezing is when he is between two and five years of age. The semen of older dogs may be successfully frozen, but often it is more sensitive to the extreme temperature changes in the process. We have successfully produced hundreds of puppies from frozen semen, many from semen frozen 20 or more years ago!!

How do I schedule an appointment?

Our freeze dates are typically the first Friday of every month with Dr. Gatlin. Please contact us below for further details.

  1. You may contact us at 508-875-7086 to speak with one of our reproductive technicians – Mary, Linn, Kirsten, or Scott. You may also schedule this appointment by contacting [email protected]
  2. Once your appointment is scheduled, please remember to bring the following paperwork:
  • AKC registration
  • a copy of current rabies vaccine certificate
  • a negative brucellosis test (if not done here).
  1. Please arrive 15-20 minutes in advance to complete paperwork and run a brucellosis test, if necessary.

How do you know if the semen is good enough to freeze?

Immediately after collection, the semen is fully evaluated. We tell you if the concentration and motility are sufficient to continue the freezing process. Good quality semen at the time of collection is essential to ensure good semen at the time of thaw and insemination. The sample is then extended with buffers that protect the sperm cells during the controlled temperature drop over four hours to -196 C. The semen is packaged into straws. The number of straws to be stored is determined by the initial sperm count. One frozen straw from your dog’s collection is thawed and examined for post-thaw motility and quality.

If your dog has not been collected in the last 6 to 12 months, we do recommend a collection and evaluation appointment prior to the freeze date to assess the quality.

When do I know the results?

At the end of the day, we will call to inform you the number of straws stored, the post-thaw motility, and the number of breedings available from the collection. We will also email you a detailed copy of the final report.

Where is the semen stored?

After your dog’s semen is collected and frozen, it safely stored here until it is shipped to Kansas City, MO, where Zoetis Inc. maintains the largest canine semen storage bank in the world. We will help establish an account for your stud dog through Zoetis’s online portal system. You will be able to access all of your frozen semen information at www.mysecurelineage.com. Once this account is established, Zoetis will contact you regarding pricing and long-term storage information. When your semen is needed, you call them to arrange for your stored semen to be shipped to the inseminating veterinarian.

The method of collecting semen, adding extender, slowly cooling, and packaging the specimen for shipment has made long distance breeding possible. We ask that you arrange for same-day or overnight semen shipment within the continental United States Monday through Saturday. 

If your stud’s semen has not been chilled before, we do recommend a pre-shipment evaluation and chill check test, although this is not required. The "chill check" is done by collecting the dog's semen and putting it through the same chilling process as if it was being shipped. We evaluate its quality every 12 hours for 48 hours. This process enables us to determine if overnight shipping is adequate or if same-day service is required.

Please fill out our chilled semen shipment form so we have all the information in advance. You may also contact our office to speak with one of our reproductive technicians.

International semen shipments require significant planning due to strict regulations. It is the client’s responsibility to research specific destination country regulations. Please contact a staff member to discuss further. You and our reproductive liaisons will need to work together to make certain that all of the regulatory requirements are met for international shipments.

Canine brucellosis is a highly contagious infection caused by a bacteria, Brucella canis. Infected dogs develop problems including infertility, spontaneous abortions, stillborn puppies, and systemic illness. Treatment is incredibly difficult and often results in euthanasia. This disease is also zoonotic, which means it can also be spread to people. Due to these concerns, we require negative brucellosis testing on all dogs and bitches every 3 months. This test can be run by most veterinary laboratories, although we do offer in-house testing, if necessary.

Kirsten evaluating semen.

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More Info

We are proud to be an AKC approved Freezing Center.  Here are some of the frequently asked questions regarding canine semen freezing:

1)   Why should I freeze my dog’s Semen?

Long Term Storage: To ensure breeding availability for future generations, you should have his semen frozen if your stud has qualities which are valuable.

For Breeding When the Stud is Not Available: Live breeding can be limited by the stud’s show or trial schedule, overbooking for the stud’s service or other scheduling conflicts.  The use of frozen semen allows availability during the bitch’s fertile time.

Long-Distance and International Breeding: Long-distance breedings may be accomplished using semen which has been either fresh-extended or frozen and eliminates the need to transport the bitch or stud dog.

2)  How is Semen collected?

Semen is collected from the stud dog by manual stimulation; the different parts or fractions of the ejaculation are collected separately, so that good quality sperm-rich semen is frozen and stored. In general, semen of better quality with higher sperm count is collected when the dog’s libido is high.  Therefore, try to closely approximate a typical breeding situation for each stud: owners are encouraged to provide a bitch in season to use as a “teaser”.  In addition, if the dog associates a particular item with breeding, such as a rug, table, breeding rack, etc., that item should be brought to the collections.

3)  What kind of paper work is needed to collect my stud?   

  • A copy of the stud’s individual registration papers.
  • Positive identification such as a tattoo or microchip.
  • Completed Semen Freeze Agreement which can be emailed to you.
  • Completed Semen Freeze Authorization which can be emailed to you.
  • A copy of the DNA Profile for your dog (which is required by AKC).  If you do not have a DNA Profile on your dog, we will submit a cheek swab sample to the AKC.
  • A copy of a negative Brucellosis test.  If your stud has not had a Brucellosis test performed then we can have the test performed the same day as the collect and freeze.  This test is a MUST in order for your males semen to be stored in our liquid nitrogen tanks.

4)  What does it cost to freeze semen?

Semen freezing is a multi-step process utilizing a variety of equipment and materials.  Please see our Reproduction price sheet in the forms section of our website for detailed information.

5)  How do I set up an appointment for Semen Freezing?

Once you have reviewed the information, please call us at 281-443-2362 or email us at [email protected] to set up an appointment.  The receptionist will take your information and our manager Jenny will return your call.  She will then discuss the process and set up the appointment with you.  We are only able to do collect and freezing on certain days so we suggest you schedule far in advance.  Dr. McGuire is our only vet that is able to perform this and she fills up quickly and can only manage at least 1- 2 freezes a day.  The collection part is easy it is the freezing that is very long and tedious.

Scheduled freezes that do not show up will not be allowed to schedule again.  Also, if your dog is unable to be collected or his semen is not viable and cannot be frozen there is still a charge for time and supplies used which is $150.

Источник: http://www.suburbiavet.com/more_info.html
dog sperm bank near me

More Info

We are proud to be an AKC approved Freezing Center.  Here are some of the frequently asked questions regarding canine semen freezing:

1)   Why should I freeze my dog’s Semen?

Long Term Storage: To ensure breeding availability for future generations, you should have his semen frozen if your stud has qualities which are valuable.

For Breeding When the Stud is Not Available: Live breeding can be limited by the stud’s show or trial schedule, overbooking for the stud’s service or other scheduling conflicts.  The use of frozen semen allows availability during the bitch’s fertile time.

Long-Distance and International Breeding: Long-distance breedings may be accomplished using semen which has been either fresh-extended or frozen and eliminates the need to transport the bitch or stud dog.

2)  How is Semen collected?

Semen is collected from the stud dog by manual stimulation; the different parts or fractions of the ejaculation are collected separately, so that good quality sperm-rich semen is frozen and stored. In general, semen of better quality with higher sperm count is collected when the dog’s libido is high.  Therefore, try to closely approximate a typical breeding situation for each stud: owners are encouraged to provide a bitch in season to use as a “teaser”.  In addition, if the dog associates a particular item with breeding, such as a rug, table, breeding rack, etc., that item should be brought to the collections.

3)  What kind of paper work is needed to collect my stud?   

  • A copy of the stud’s individual registration papers.
  • Positive identification such as a tattoo or microchip.
  • Completed Semen Freeze Agreement which can be emailed to you.
  • Completed Semen Freeze Authorization which can be emailed to you.
  • A copy of the DNA Profile for your dog (which is required by AKC).  If you do not have a DNA Profile on your dog, we will submit a cheek swab sample to the AKC.
  • A copy of a negative Brucellosis test.  If your stud has not had a Brucellosis test performed then we can have the test performed the same day as the collect and freeze.  This test is a MUST in order for your males semen to be stored in our liquid nitrogen tanks.

4)  What does it cost to freeze semen?

Semen freezing is a multi-step process utilizing a variety of equipment and materials.  Please see our Reproduction price sheet in the forms section of our website for detailed information.

5)  How do I set up an appointment for Semen Freezing?

Once you have reviewed the information, please call us at 281-443-2362 or email us at [email protected] to set up an appointment.  The receptionist will take your information and our manager Jenny will return your call.  She will then discuss the process and set up the appointment with you.  We are only able to do collect and freezing on certain days so we suggest you schedule far in advance.  Dr. McGuire is our only vet that is able to perform this and she fills up quickly and can only manage at least 1- 2 freezes a day.  The collection part is easy it is the freezing that is very long and tedious.

Scheduled freezes that do not show up will not be allowed tioga state bank newfield schedule again.  Also, if your dog is unable to be collected or his semen is not viable and cannot be frozen there is still a charge for time and supplies used which is $150.

Источник: http://www.suburbiavet.com/more_info.html

Independent and combined effects of diethylhexyl phthalate and polychlorinated biphenyl 153 on sperm quality in the human and dog

Abstract

A temporal decline in human and dog sperm quality is thought to reflect a common environmental aetiology. This may reflect direct effects of seminal chemicals on sperm function and quality. Here we report the effects of diethylhexyl phthalate (DEHP) and polychlorinated biphenyl 153 (PCB153) on DNA fragmentation and motility in human and dog sperm. Human and dog semen was collected from registered donors (n = 9) and from stud dogs (n = 11) and incubated with PCB153 and DEHP, independently and combined, at 0x, 2x, 10x and 100x dog testis concentrations. A total of 16 treatments reflected a 4 × 4 factorial experimental design. Although exposure to DEHP and/or PCB153 alone increased DNA fragmentation and decreased motility, the scale of dose-related effects varied with the presence and relative concentrations of each chemical (DEHP.PCB interaction for: DNA fragmentation; human p < 0.001, dog p < 0.001; Motility; human p < 0.001, dog p < 0.05). In both human and dog sperm, progressive motility negatively correlated with DNA fragmentation regardless of chemical presence (Human: P < 0.0001, r = −0.36; dog P < 0.0001, r = −0.29). We conclude that DEHP and PCB153, at known tissue concentrations, induce similar effects on human and dog sperm supporting the contention of the dog as a sentinel species for human exposure.

Introduction

Over the last four decades, there has been increasing concern over declining human male reproductive health. Reduced sperm counts have been widely used as an index of male subfertility and meta-analytical studies indicate a 50% global reduction in quality from 1938 to 20111,2,3. Sperm morphology has also been reported to decrease over a period of 17 years in France with some geographical regional variation; Aquitaine and Midi-Pyrenees having the lowest morphology combined with the lowest concentrations4,5. These data are indicative of an environmental aetiology and, in support of this, epidemiological studies showing increased incidences of testicular cancer and malformations at birth have been linked to regions with reduced sperm counts6,7.

Temporal trends in human semen quality are paralleled by a similar trend in dogs that live in the human household, where sperm motility declined by 30% over a 26 year period8. In this latter study, all data was generated from a single laboratory using consistent techniques and thus did not suffer from changes in methodology and quality assurance over the time span encompassed in human meta-analytical studies9. These observations support the hypothesis that temporal trends in semen quality, both in the human and dog, are due to shared environmental factors and that the dog may be a sentinel for human exposure to such factors. Access to a controlled breeding population of assistance dogs that are routinely sampled for sperm quality provides a cost-effective means of sperm analyses without the stigma and social complications that accompany analogous human studies. Furthermore, there is considerable potential to extend these analyses to any individual or population of dogs. For example, although not investigated in the current study, this could be achieved by semen collection from the tail of the epididymis10 immediately after removal of dog testes at routine surgical neutering; a procedure that is performed on hundreds of thousands of dogs worldwide each year. In addition, semen collections from live dogs is a procedure that is tolerated by a majority of breeds11 unaccustomed to routine fertility monitoring and can therefore easily be carried out by a trained technician.

Declining sperm quality emb agar ingredients been linked with the exposure to persistent anthropogenic chemicals, many of which exhibit endocrine disrupting activity6. Although the mechanisms underlying these putative effects are uncertain, historically, the period of fetal development has been highlighted as being particularly sensitive to chemicals with endocrine disrupting activity12. However, a number of studies have shown that environmental chemicals (ECs) are present in semen in a range of species, including the human, raising the possibility of a direct acute effect of chemicals on sperm13,14,15. In support of this theory, an elevated concentration of seminal bisphenol A (BPA) has been associated with infertility in men15,16 and elevated human seminal phthalate metabolites have been associated with reduced sperm counts17. In a separate study, the phthalates DEHP and di-n-butyl-phthalate (DBP) in human semen were reported to be inversely associated with motility and this was confirmed by the direct application of the same phthalates, at seminal concentrations, to sperm in vitro18. By contrast, PCB congeners 118, 126 and 153 were reported to have no negative effect on human sperm motility in vitro, both individually and in combination19. Similar findings have been reported in the dog where DEHP and PCB153, at concentrations detected in dog semen and testis, exhibited inhibitory and stimulatory effects respectively when tested on sperm motility in vitro8. In the same study, both DEHP and PCB153 were detected in a range of dry and wet dog foods indicative of a dietary source. Both DEHP and PCB153 are widely present in the environment and have been detected in tissues/fluids ranging from human breast milk to ovine liver. DEHP is a widely used plasticizer that leaches out into food and liquids and PCBs are lipophilic, and are therefore present in fatty foods20,21,22. Consequently, exposure occurs largely through the diet and these chemicals are deemed as risk factors for reproductive function23,24,25. In support of this, our own published study has shown that DEHP, PCB153 and other PCB congeners are present within both dry and wet dog food sources8 [DEHP: wet and dry food, 0.37 ± 0.10 and 0.20 ± 0.03 μg/g respectively; ∑PCBs: wet and dry food, 1.35 ± 0.5792 and 0.78 ± 0.223 μg/kg respectively; PCB153: wet and dry food, 0.39 ± 0.193 and 0.22 ± 0.11 μg/kg respectively].

Another parameter of ejaculate quality is the proportion of sperm that exhibit DNA fragmentation26. Environmental chemicals have been shown to induce both human and dog sperm DNA fragmentation8,27 and a commercial mixture of PCBs (Arochlor), administered to rats in vivo and added to sperm in vitro, also increased sperm DNA fragmentation28.

In total, these data suggest that environmental chemicals induce similar acute effects on human and dog sperm in vitro: measurement of sperm motility and DNA fragmentation are tried and tested measures of such chemical effects. Since sperm concentration would not alter during the period of in vitro culture and morphology is confounded by abnormality classification, partly due to swelling that may occur during culture and the generation of artefacts during processing, these parameters were not selected for testing acute chemical effects in vitro29,30. Notwithstanding, testing the effects of individual chemicals on sperm functional parameters does not represent “real-life” exposure to a mixture of chemicals, many of which exhibit synergistic, antagonistic or additive effects. Two chemicals known to be present in dog seminal plasma and testis were therefore selected and their effects tested both independently and in combination, on sperm motility and DNA fragmentation in both the human and dog.

Results

Chemical effects on percentage normal sperm motility in human and dog

The analyses of both the human and the dog sperm motility data found the interaction terms between PCB and DEHP to be significant (human p < 0.001; dog p < 0.05). This indicates that the dose response to either chemical was not independent of the level of the other chemical dog sperm bank near me. Figure 1 illustrates the effects of PCB153 and DEHP, individually and combined, on dog and human sperm motility. In dog sperm, PCB153 in the absence of DEHP induced a dose-dependent inhibitory effect on sperm motility (Fig. 1ai). In the presence of DEHP at 2x and 10x mean testis concentration (MTC), no dose dependent inhibitory effect of PCB153 was observed. However, reduced motility was observed in the presence of 100x DEHP co-incubated with 2x and 10x PCB153 (Fig. 1aii).

Effect of DEHP and PCB153 on dog and human sperm motility. Chemicals tested individually [ai,bi: PCB only; aii,bii: grey bars: DEHP only] and in combination [aii,bii]. Graphs display fixed concentrations of DEHP with increasing concentrations of PCB153 in dog [ai,aii: p <  0.01] and human [bi,bii: p <  0.001] sperm. Grade a motility: >25 μm/s Error bar = 1 Standard Error of Difference. MTC = Mean testis concentration.

Full size image

In contrast to the dog, neither PCB153 nor DEHP, in the absence of the other chemical, influenced human sperm motility (Fig. 1bi,bii). However, in the presence of 2x DEHP, a dose dependent inhibitory effect in response to PCB153 was observed (Fig. 1bii). The inhibitory effect of PCB153 was broadly maintained in the presence of 10x and 100x DEHP with the exception of 10x PCB153/10x DEHP and 100x PCB153/100x DEHP, where no inhibition was observed (Fig. 1bii).

Chemical effects on sperm DNA integrity in human and dog

For both the human and dog DNA fragmentation data, significant (P < 0.001) PCB.DEHP interaction terms were found. Again, this indicates that the response of DNA integrity to one chemical is not independent of the presence or level of the other chemical. Figure 2 illustrates the effects of PCB153 and DEHP, individually and combined, on both dog and human sperm DNA fragmentation using the sperm chromatin dispersion assay. In the dog, PCB153 in the absence of DEHP induced a dose-dependent increase in sperm DNA fragmentation (Fig. 2ai). A similar dose-dependent increase in DNA fragmentation was observed in response to DEHP in the absence of PCB153 (Fig. 2aii). When PCB153 and DEHP were tested in combination, DNA fragmentation was still increased at higher concentrations of PCB153 but the dose-dependent response was blunted (Fig. 2aii). The response of human sperm to PCB153 and DEHP independently and combined, paralleled that observed in the dog. Figure 2b illustrates that human sperm incubated with PCB153 or DEHP in the absence of the other chemical, exhibited a dose-dependent increase in DNA fragmentation (Fig. 2bi,bii). In addition, DNA fragmentation was increased in response to both chemicals tested in combination although additive effects were not apparent.

Effect of DEHP and PCB153 on dog and human sperm DNA fragmentation. Chemicals tested individually [ai,bi: PCB only; aii,bii: grey bars: DEHP only] and in combination [aii,bii]. Graphs display fixed concentrations of DEHP with increasing concentrations of PCB153 in dog [ai,aii: p ≤ 0.001] and human [bi,bii: p ≤ 0.001] sperm. Error bar = 1 Standard Error of difference between means. MTC = Mean testis concentration.

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Sperm DNA fragmentation and motility correlations

Figure 3 illustrates the relationship between progressive motility and DNA fragmentation in the presence and absence of each chemical independently and combined. Despite chemical effects on sperm motility (Fig. 1) and DNA fragmentation (Fig. 2), the relationship between these two parameters remained the same regardless of the nature of the chemical exposure. All correlations were significant except the human control samples [Dog (Fig. 3i, n = 352): Control; p < 0.05, r = −0.527, n = 22; DEHP; p < 0.05, r = −0.2862, n = 66; PCB-153; p < 0.01, r = −0.3276, n = 66; Mixture; p < 0.0001, r = −0.2826, n = 198; vs Human (Fig. 3ii, n = 288): Control; p > 0.05, r = −0.4374, n = 18; DEHP; p < 0.05, county bank routing number, n = 54; PCB-153; p < 0.05, r = −0.2994, n = 54; Mixture; p < 0.0001, r = −0.4037, n = 162].

Correlation between progressive motility and DNA fragmentation in both dog and human sperm. Values from 32 sperm assessments (16 treatments, two time points). Each point represents a different sperm culture equating to a total n = 352 [dog] and n = 288 [human]. Colours denote culture media constituents to demonstrate spread: Control (black), DEHP (red), PCB153 (blue) and mixture (green). Dog (i, n = 352): Control; p < 0.05, r = −0.527, n = 22; DEHP; p < 0.05, r = −0.2862, n = 66; PCB153; p < 0.01, r = −0.3276, n = 66; Mixture; p < 0.0001, r = −0.2826, n = 198; vs Human (ii, n = 288): Control; p > 0.05, r = −0.4374, n = 18; DEHP; p < 0.05, r = −0.3118, n = 54; PCB153; p < 0.05, r = −0.2994, n = 54; Mixture; p < 0.0001, r = −0.4037, n = 162]. Confidence bands (95%) plotted for visual purposes only. Progressive motility based on WHO pre-2010 where sperm swimming grades a and b are combined: ≥5 µm/s.

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Discussion

Data presented in this paper are significant because we conclusively demonstrate that a selected phthalate (DEHP) and PCB congener (PCB153), at concentrations relevant to environmental exposure, reduce dog sperm motility and increase DNA fragmentation in vitro. In the human, our data showing reduced motility and increased DNA fragmentation are indicative of similar sensitivities to these chemicals in both species. Furthermore, when the chemicals were combined and co-incubated with dog or human sperm, the overall impact on motility or DNA fragmentation was dependent on the concentration ratio. This is the first study to select two environmental chemicals and to test them at four concentrations in all possible combinations representative of those found in the male reproductive tract and fluids. In addition, a negative correlation between motility and DNA fragmentation, that incorporates chemical variables, is described in both species.

Our data on the sensitivity of sperm to short term chemical exposure support similar studies using a range of environmentally relevant chemical challenges in vitro. For example, DEHP, DBP and mono-n-butyl phthalate (MBP), are reported to reduce sperm motility in vitro when added at seminal concentrations measured in infertile men18,31. A mixture of PCBs are also reported to reduce sperm motility in both the human and pig32,33 and in the human, p,p’-dichloro-diphenyl-dichloro-ethylene (p,p’-DDE) has been shown to increase Ca2+ uptake by sperm in a mechanism that involves the CatSper channel34.

To minimise the effects of pre-exposure to chemicals present in ejaculates collected for culture, voopoo finic fish current study used a concentration range that encompassed the reported variability in seminal concentrations and reduced pre-existing chemical concentrations by sperm processing and washing prior to culture. The subsequent comparison to controls, with no further chemical added, provided a means of testing chemical effects. Despite these steps, pre-exposure differences will inevitably exist between, and within, species. For example, in the current study, when DEHP is present at twice the concentration found in testis, dog sperm appears to be less sensitive to lower PCB concentrations than human sperm. In populations of men from Greenland, Sweden, Poland and the Ukraine, increased seminal PCB153 has been consistently associated with reduced sperm motility but not concentration or morphology35,36. In addition, sperm DNA fragmentation was positively associated with PCB153 in European populations but not in samples from Greenlandic men: an observation that likely reflects different pre-exposures.

In the current study, our intention was not to measure and equate chemical concentrations in each individual sample with sperm motility and fragmentation, but to use a concentration range previously established in the dog as a ballpark estimate of chemical concentrations in the male reproductive tract8. That is, concentrations found in the dog reproductive tract and seminal plasma, used as an indicator of those likely present in the human. In support of this contention, human seminal PCB (total) and DEHP concentrations reported in populations of fertile men [total PCB: up to 5.8 ng/ml, DEHP: mean of 0.61 μg/ml]37,38 are comparable to those detected in the dog [PCB: 0.26–13.2 ng/ml; DEHP: 0.75–37.5 μg/ml]8.

Notably, elevated concentrations of both chemicals have been linked with reduced human male fertility18. Semen PCB concentrations have been reported to be higher in a population of ‘infertile’ men (parameters stated as <20 million/ml or <25% progressive motility and/or <30% normal morphology)25. Further, a research group in China reported an association between increased urinary phthalate metabolites, reduced sperm count and increased sperm DNA damage14. Although the mechanism was not proven, the authors suggest that this likely reflects chemical effects on testicular Sertoli and germ cells as reported in animal studies39,40,41,42.

Although both DEHP and PCB153 have been reported to reduce sperm motility and increase sperm DNA fragmentation individually in the human8,18,37, they have not been assessed in combination, at environmentally relevant concentrations, as reported here. A limited number of studies have investigated some combinations of environmental chemicals for their effect on human sperm function32. Real-life exposure is to a complex mixture of chemicals that are likely to exhibit synergistic or antagonistic, as well as additive effects, on sperm. Mixtures of endocrine disrupting chemicals have been shown to cooperatively increase Ca2+ concentrations in sperm through the activation of the principle calcium channel CatSper43.

In the current study, the mechanisms that underlie the concentration ratio-dependent effects of the two chemicals combined remain uncertain. PCBs and DEHP are considered to be pro-estrogenic and anti-androgenic respectively raising the possibility that a change in concentration ratio may alter the relative activation of sperm estrogen or androgen receptors44,45. Indeed, in a population of ‘infertile’ men (parameters stated as sperm count < 20 × 106, motility < 50%, morphology < 14% normal) androgen receptor expression is reported to positively correlate with sperm motility46 and PCBs are reported to affect sperm concentration and motility relative to the number of CAG repeats in the androgen receptor gene47. This may account for PCB influences on motility even in the presence of DEHP. Estrogenic compounds have also been reported to reduce human sperm motility in vitro via an induction in redox activity and this mechanism has also been linked to the induction of DNA fragmentation by 2-hydroxy estradiol48.

The greater consistency of chemical effects on human and dog sperm DNA fragmentation compared to motility is interesting and emphasises the importance of looking at more than one sperm functional/viability parameter when assessing environmental effects. It is important to note however that despite this subtle difference, the two sperm parameters were highly correlated in both species and remained so in the presence of the chemicals independently and combined. This is an important observation since it has been reported that human male infertility is associated with increased levels of sperm DNA damage and that sperm motility defects are highest in samples with increased DNA fragmentation49,50. This raises the possibility that there may be a similar relationship between sperm DNA fragmentation, motility and fertility in the dog.

In conclusion, we have demonstrated that the use of low dose tissue relevant concentrations of DEHP and PCB153, independently and in combination, negatively impact on christian financial credit union chesterfield michigan motility and DNA fragmentation in samples obtained from humans and dogs. Since these effects are broadly similar in both species, this raises the possibility that the environmental impact of chemicals in the dog may provide a means of investigating pollutant effects on mammalian fertility in a species in which external influences, such as diet, are better controlled than in an equivalent human study.

Methods

Ethical Approval

Human

Semen donations were obtained from anonymous HFEA registered donors (n = 9) attending the fertility unit at Nottingham University Hospitals. Donors provided informed consent for the use of samples in this research project ensuring ‘General Data Protection Regulation’ compliance. Each donor was initially screened following HFEA and British Fertility Society protocols51. No samples from fertility patients were used and all donors completed HFEA consent forms. Ethical approval was obtained from the School of Veterinary Medicine Ethical Review Committee [Reference 1511,150723]. The HFEA consent forms completed by donors and ethical approval documents were also approved by the Chair of the Ethical Review Committee of the Faculty of Medicine and Health Sciences, University of Nottingham. In accordance with the Royal College of Pathologists guidance on the use of pathological specimens [https://www.rcpath.org/resourceLibrary/the-retention-and-storage-of-pathological-records-and-specimens-5th-edition.html], no further ethical approval was required.

Dog

Semen was collected as part of routine reproductive examination of stud dogs subject to owner consent with full GDPR compliance. Due to dog sperm being collected as part of routine reproductive health checks, the economical outlay associated with sample collection was minimised. The dogs resided in the same region of the UK and lived in the modern household with owners briefed on use of controlled diet and exercise regimes. All semen collections were performed in accordance with relevant guidelines and regulations and the collection protocol was approved by the School of Veterinary Medicine Ethical Review Committee (Refs: 208 101012, 513 120117 and 1097 140227).

Human sperm collection and preparation

Samples were liquefied for 20 minutes at room temperature prior to semen preparation. Ejaculate underwent density gradient centrifugation using a Universal 320 R Hettich centrifuge (DJB Labcare, Newport Pagnell, UK) at 25 °C. Briefly, one millilitre of 40% isotonic density gradient was loaded onto one millilitre of 80% isotonic medium [PureSperm 40/80, Nidacon, Sweden]. Two millilitres of liquefied semen were loaded onto both isotonic mediums and reagents centrifuged at 300 × g for 22 minutes. On completion, the sperm rich pellet was re-suspended using 1.5 ml PureSperm wash (pH range of 7.3–8.5; osmolality: 290–300 mOsm/kg H2O, Nidacon, Sweden).

Dog sperm collection

Ejaculate was collected from stud dogs (n = 11) by routine digital manipulation. The sperm rich fraction (fraction 2) was collected into sterile plastic 15 millilitre Greiner centrifuge tubes [Sigma-Aldrich, Dorset, UK]. INRA extending medium [INRA, Nouzilly, France] was added to sperm at a 2:1 ratio to aid sperm survival.

Chemical preparation

The two chemicals selected for co-culture with sperm were diethylhexyl phthalate (DEHP) and polychlorinated biphenyl congener 153 (PCB153) and concentrations were calculated relative to those present in dog testicular tissue8. The rationale for this was (1) dog testis concentrations of both chemicals have been established and standardised in our previous study and are reflective of exposure of the male reproductive tract8 (2) dog and human semen PCB concentrations are variable but generally higher than testis and in range of the concentrations tested in vitro8,25,52 (3) although dog semen DEHP concentrations have not been determined, due to the large amount of dry material required, reported human DEHP concentrations are in range of established dog testis measurements14,18,53. On this basis, dog testis concentrations were used as a standardised measure of environmental exposure relevant to both species. DEHP (CAS no: 117-81-7) and PCB153 (CAS no: 35065-27-1) [Sigma-Aldrich, Dorset, U.K.] were dissolved in 100% dimethylsulphoxide (DMSO) and diluted with PBS to 4x, 20x and 200x mean testis concentration containing 0.02% DMSO. Chemical preparations were co-incubated with sperm at a 1:1 ratio with a final exposure concentration of 2x, 10x and 100x mean testis concentration. Sperm were incubated with each chemical at each concentration individually and with a mixture of the two chemicals in all 16 combination ratios (Table 1). Acute treatment effects were assessed at 10 minutes and 3 hours and control incubations carried out with 0.01% DMSO only.

Full size table

Motility assessment

Sperm motility was assessed using Computer Assisted Sperm Analysis (CASA) software. Sperm were acclimatised for two minutes on a 37 °C stage prior to assessment. Five µl of sperm, irrespective of species, were pipetted into a specialised 20 µl cellvision glass slide counting chamber (Code CV1020-2CV; CellVision, the Netherlands). A minimum of 200 sperm were the huntington national bank inc for each treatment. For human sperm, motility was tracked by use of the diagnostic software ‘SAMi’ (Procreative Diagnostics Ltd, Staffordshire, UK) and an Olympus BH2 Microscope [KeyMed (Medical and Discover bank custodial account Equipment) Ltd, Essex, UK]. Dog sperm motility was assessed using the Hobson’s CASA tracking system (Hobson’s tracking systems Ltd., Sheffield, UK) and viewed using a negative-high phase contrast objective (x20) on an Olympus BH2 microscope fitted with a camera ocular. For both species, sperm motility was assessed according to WHO 199954 where grade a motility was classified as ≥ 25 µm/s and progressive motility ≥ 5 µm/s (grades a & b combined).

DNA fragmentation

Sperm DNA fragmentation was quantified by defragmentation index (sDFI %) measured using the sperm chromatin dispersion assay55. Sperm were assessed based on the size of the halos, indicative of sperm that exhibited nuclei with non-fragmented DNA. Sperm with denatured or fragmented DNA were identified by the absence of a halo, or a halo that was exceedingly small. Briefly, equal volumes of intact, unfixed sperm and 1% agarose solution were combined. Fifteen microliter aliquots of sperm-agarose suspensions were pipetted onto agarose pre-coated poly-L-lysine slides (CAS: P4981; ThermoFisher Scientific Ltd. UK), covered with a 22 mm × 22 mm cover-slip, and placed in a 4 °C environment for five minutes to fix the sample. After an acid treatment (0.08 M HCl) of seven minutes, sperm were incubated in a lysing reagent (0.8 M DTT, 0.4 M Tris, 2 M NaCl, 1% triton-X) for 20 minutes. Slides were then submerged in dH2O for a period of five minutes followed by dehydration through a series of ethanol solutions (70%, 90% and 100% respectively). Visualisation was obtained using Diff-Quick staining reagents (eosinophilic and basophilic stains; CAS 9990700, ThermoFisher Scientific Ltd, Paisley, UK) and analysis undertaken using oil immersion, at 1000x magnification [Leica DM 5000 B microscope; Leica Microsystems, Milton Keynes, UK]. A minimum of 200 sperm were assessed for each treatment. Positive controls were incubated with 300 µm H2O2 before denaturation and negative controls by omission of the denaturation step. To determine the defragmentation index, the number of fragmented sperm was divided by the total number of sperm counted. This provided a value for the proportion of sperm that were fragmented. This proportion was then plotted on the logit scale.

Experimental design and Statistical analysis

For both the human and dog experiments, sperm samples were treated with PCB153 at one of four concentrations in combination with DEHP, also at one of four concentrations. The concentrations used for both chemicals corresponded to 0x, 2x, 10x and 100x the baseline concentration measured from dog testes. Thus there were 16 possible combinations of PCB153 and DEHP. Each combination was randomly allocated to one of 16 separate sub-samples of sperm, from each replicate donor.

Statistical analysis was undertaken using GenStat 17th edition (VSN International Ltd, Hempstead, UK). The proportions out of known numbers of sperm that had a particular characteristic were analysed as grouped binary data by fitting a generalised linear mixed model with a logit link function, assuming a binomial error distribution. The fixed effects included in the statistical model were PCB, DEHP and the PCB.DEHP interaction term. Another fixed effect, TIME, and its interaction terms were included when two repeated samples from the same tube were measured three hours apart. The random effects were donor and culture-tube within donor.

The statistical significance of a fixed effect was tested using an F-Ratio test. The analysis output provided predicted mean proportions and standard errors of difference between means which were graphically represented on a logit scale. Logit values were converted back into proportions and graphically plotted [GraphPad Prism 7.0, GraphPad Ltd, California, USA]. A single error bar on each figure represents the standard error of the difference between means. Where correlations were investigated, data was assumed to be non-normally distributed and a Spearman’s rank correlation analysis was undertaken to provide correlation coefficients between motility and DNA fragmentation.

Data Availability

The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

References

  1. 1.

    Carlsen, E., Giwercman, A., Keiding, N. & Skakkebaek, N. E. Evidence for decreasing quality of semen during past 50 years. BMJ305, 609–613 (1992).

    CASArticle Google Scholar

  2. 2.

    Swan, S. H., Elkin, E. P. & Fenster, L. The question of declining sperm density revisited: an analysis of 101 studies published 1934–1996. Environ. Health Perspect.108, 961–966 (2000).

    CASArticle Google Scholar

  3. 3.

    Levine, H. et al. Temporal trends in sperm count: a systematic review and meta-regression analysis. Hum. Reprod. Update23, 646–659 (2017).

    Article Google Scholar

  4. 4.

    Rolland, M., Le Moal, J., Wagner, V., Royere, D. & De Mouzon, J. Decline in semen concentration and morphology in a sample of 26,609 men close to general population between 1989 and 2005 in France. Hum. Reprod. 28, 462–470 (2013).

  5. 5.

    Le Moal, J. et al. Semen quality trends in French regions are consistent with a global change in environmental exposure. Reproduction147, 567–574 (2014).

    Article Google Scholar

  6. 6.

    Bay, K., Asklund, C., Skakkebaek, N. E. & Andersson, A.-M. Testicular dysgenesis syndrome: possible role of endocrine disrupters. Best Pract. Res. Clin. Endocrinol. Metab.20, 77–90 (2006).

    CASArticle Google Scholar

  7. 7.

    Skakkebaek, N. E. Sperm counts, testicular cancers, and the environment. BMJ (Clinical research ed.)359, j4517 (2017).

    Article Google Scholar

  8. 8.

    Lea, R. G. et al. Environmental chemicals impact dog semen quality in vitro and may be associated with a temporal decline in sperm motility and increased cryptorchidism. Sci. Rep.6, 31281 (2016).

    ADSCASArticle dog sperm bank near me Google Scholar

  9. 9.

    Pacey, A. A. Are sperm counts declining? Or did we just change our spectacles? Asian J. Androl.15, 187–190 (2013).

    Article Google Scholar

  10. 10.

    Ponglowhapan, S., Chatdarong, K., Sirivaidyapong, S. & Lohachit, C. Freezing of epididymal spermatozoa from dogs after cool storage for 2 or 4 days. Theriogenology66, 1633–1636 (2006).

    CASArticle Google Scholar

  11. 11.

    Hermansson, U. & Linde Forsberg, C. Freezing of stored, chilled dog spermatozoa. Theriogenology65, 584–593 (2006).

    Article Google Scholar

  12. 12.

    Lea, R. G. et al. Endocrine disruptors and ovine reproductive development. In Reproduction in Domestic Ruminants Vol.VIII (eds Juengel, J. et al.) 209–227 (Context Products Ltd., UK, 2014).

  13. 13.

    Kamarianos, A., Karamanlis, X., Theodosiadou, E., Goulas, P. & Smokovitis, A. The presence of environmental pollutants in the semen of farm animals (bull, ram, goat, and boar). Reprod. Toxicol.17, 439–445 (2003).

    CASArticle Google Scholar

  14. 14.

    Wang, Y.-X. et al. Phthalate exposure and human semen quality: Results from an infertility clinic in China. Environ. Res.142, 1–9 (2015).

    ADSCASArticle Google Scholar

  15. 15.

    La Rocca, C. et al. Exposure to Endocrine Disruptors and Nuclear Receptors Gene Expression in Infertile and Fertile Men from Italian Areas with Different Environmental Features. Int. J. Environ. Res. Public Health12, 12426–12445 (2015).

    Article Google Scholar

  16. 16.

    Vitku, J. et al. Difflerences in bisphenol A and estrogen levels in the plasma and seminal plasma of men with different degrees of infertility. Physiol. Res.64(Suppl 2), S303–11 (2015).

    CASPubMed Google Scholar

  17. 17.

    Chang, W.-H., Wu, M.-H., Pan, H.-A., Guo, P.-L. & Lee, C.-C. Semen quality and insulin-like factor 3: Associations with urinary and seminal levels of phthalate metabolites in adult males. Chemosphere173, 594–602 (2017).

    ADSCASArticle Google Scholar

  18. 18.

    Pant, N. et al. Environmental and experimental exposure of phthalate esters: the toxicological consequence on human sperm. Hum. Exp. Toxicol.30, 507–514 (2011).

    CASArticle Google Scholar

  19. 19.

    Pflieger-Bruss, S. et al. Effects of single non-ortho, mono-ortho, and di-ortho chlorinated biphenyls on human sperm functions in vitro. Reprod. Toxicol.21, 280–284 (2006).

    CASArticle Google Scholar

  20. 20.

    Kastner, J., Cooper, D. G., Maric, M., Dodd, P. & Yargeau, V. Aqueous leaching of di-2-ethylhexyl phthalate and ‘green’ plasticizers from poly(vinyl chloride). Sci. Total Environ.432, 357–364 (2012).

    ADSCASArticle Google Scholar

  21. 21.

    Erythropel, H. C., Maric, M., Nicell, J. A., Leask, R. L. & Yargeau, V. Leaching of the plasticizer di(2-ethylhexyl)phthalate (DEHP) from plastic containers and the question of human exposure. Appl. Microbiol. Biotechnol.98, 9967–9981 (2014).

    CASArticle Google Scholar

  22. 22.

    Faroon, O. & Ruiz, P. Polychlorinated biphenyls: New evidence from the last decade. Toxicol. Ind. Health32, 1825–1847 (2016).

    CASArticle Google Scholar

  23. 23.

    Pocar, P. et al. Exposure to di(2-ethyl-hexyl) phthalate (DEHP) in utero and during lactation causes long-term pituitary-gonadal axis disruption in male and female mouse offspring. Endocrinology153, 937–948 (2012).

    CASArticle Google Scholar

  24. 24.

    Oskam, I. C. et al. Effects of long-term maternal exposure to low doses of PCB126 and PCB153 on the reproductive system and related hormones of young male goats. Reproduction130, 731–742 (2005).

    CASArticle Google Scholar

  25. 25.

    Rozati, R., Reddy, P. P., Reddanna, P. & Mujtaba, R. Role of environmental estrogens in the deterioration of male factor fertility. Fertil. Steril.78, 1187–1194 (2002).

    Article Google Scholar

  26. 26.

    Rex, A. S., Aagaard, J. & Fedder, J. DNA fragmentation in spermatozoa: a historical review. Andrology5, 622–630 (2017).

    CASArticle Google Scholar

  27. 27.

    Evenson, D. P. & Wixon, R. Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA). Toxicol. Appl. Pharmacol.207, 532–537 (2005).

    Article Google Scholar

  28. 28.

    Aly, H. A. A. Aroclor 1254 induced oxidative stress and mitochondria mediated apoptosis in adult rat sperm in vitro. Environ. Toxicol. Pharmacol.36, 274–283 (2013).

    CASArticle Google Scholar

  29. 29.

    Mortimer, D. & Menkveld, R. Sperm morphology assessment—historical perspectives and current opinions. J. Androl.22, 192–205 (2001).

    CASPubMed Google Scholar

  30. 30.

    Boersma, A., Rasshofer, R. & Stolla, R. Influence of sample preparation, staining procedure and analysis conditions on bull sperm head morphometry using the morphology analyser integrated visual optical system. Reprod. Domest. Anim.36, 222–229 (2001).

    CASArticle Google Scholar

  31. 31.

    Xie, F. et al. Effects of two environmental endocrine disruptors di-n-butyl phthalate (DBP) and mono-n-butyl phthalate (MBP) on human sperm functions in vitro. Reprod. Toxicol.83, 1–7 (2019).

    CASArticle Google Scholar

  32. 32.

    Jiang, L.-G. et al. Toxic effects of polychlorinated biphenyls (Aroclor 1254) on human sperm motility. Asian J. Androl.19, 561–566 (2017).

    CASArticle Google Scholar

  33. 33.

    Campagna, C., Guillemette, C., Ayotte, P. & Bailey, J. L. Effects of an environmentally relevant organochlorine mixture and a metabolized extract of this mixture on porcine sperm parameters in vitro. J. Androl.30, 317–324 (2009).

    CASArticle Google Scholar

  34. 34.

    Tavares, R. S., Escada-Rebelo, S., Correia, M., Mota, P. C. & Ramalho-Santos, J. The non-genomic effects of endocrine-disrupting chemicals on mammalian sperm. Reproduction151, R1–R13 (2016).

    CASArticle Google Scholar

  35. 35.

    Toft, G. Persistent organochlorine pollutants and human reproductive health. Dan. Med. J.61, B4967 (2014).

    PubMed Google Scholar

  36. 36.

    Toft, G. et al. Semen quality and exposure to persistent organochlorine pollutants. Epidemiology17, 450–458 (2006).

    Article Google Scholar

  37. 37.

    Bush, B., Bennett, A. H. & Snow, J. T. Polychlorobiphenyl congeners, p,p’-DDE, and sperm function in humans. Arch. Environ. Contam. Toxicol.15, 333–341 (1986).

    CASArticle Google Scholar

  38. 38.

    Han, S. W. et al. An exposure assessment of di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) in human semen. J. Toxicol. Environ. Heal. Part A72, 1463–1469 (2009).

    CASArticle Google Scholar

  39. 39.

    Jones, S., Boisvert, A., Francois, S., Zhang, L. & Culty, M. In utero exposure to di-(2-ethylhexyl) phthalate induces testicular effects in neonatal rats that apply for emergency food stamps nyc antagonized by genistein cotreatment. Biol. Reprod.93(92), 1–14 (2015).

    CAS Google Scholar

  40. 40.

    Richburg, J. H. & Boekelheide, K. Mono-(2-ethylhexyl) phthalate rapidly alters both Sertoli cell vimentin filaments and germ cell apoptosis in young rat testes. Toxicol. Appl. Pharmacol.137, 42–50 (1996).

    CASArticle Google Scholar

  41. 41.

    Ungewitter, E. et al. From the Cover: Teratogenic Effects of in Utero Exposure to Di-(2-Ethylhexyl)-Phthalate (DEHP) in B6:129S4 Mice. Toxicol. Sci.157, 8–19 (2017).

    CASPubMedPubMed Central Google Scholar

  42. 42.

    Zhang, T. et al. Dog sperm bank near me protects prepuberal testis from deleterious effects of bisphenol A or diethylhexyl phthalate by preserving H3K9 methylation. J. Pineal Res.65, e12497 (2018).

    Article Google Scholar

  43. 43.

    Schiffer, C. et al. Direct action of endocrine disrupting chemicals on human sperm. EMBO Rep.15, 758–765 (2014).

    CASArticle Google Scholar

  44. 44.

    Aquila, S. et al. Estrogen receptor (ER)alpha and ER beta are both expressed in human ejaculated spermatozoa: evidence of their direct interaction with phosphatidylinositol-3-OH kinase/Akt pathway. J. Clin. Endocrinol. Metab.89, 1443–1451 (2004).

    CASArticle Google Scholar

  45. 45.

    Aquila, S. et al. Human sperm express a functional androgen receptor: effects on PI3K/AKT pathway. Hum. Reprod.22, 2594–2605 (2007).

    CASArticle Google Scholar

  46. 46.

    Zalata, A. A. et al. Androgen receptor expression relationship with semen variables in infertile men with varicocele. J. Urol.189, 2243–2247 (2013).

    CASArticle Google Scholar

  47. 47.

    Giwercman, A., Rylander, L. & Lundberg Giwercman, Y. Influence of endocrine disruptors on human male fertility. Reprod. Biomed. Online15, 633–642 (2007).

    CASArticle Google Scholar

  48. 48.

    Bennetts, L. E. et al. Impact of estrogenic compounds on DNA integrity in human spermatozoa: evidence for cross-linking and redox cycling activities. Mutat. Res.641, 1–11 (2008).

    ADSCASArticle Google Scholar

  49. 49.

    Velez de la Calle, J. F. et al. Sperm deoxyribonucleic acid fragmentation as assessed by the sperm chromatin dispersion test in assisted reproductive technology programs: results of a large prospective multicenter study. Fertil. Steril.90, 1792–1799 (2008).

    Article Google Scholar

  50. 50.

    Belloc, S. et al. Sperm deoxyribonucleic acid damage in normozoospermic men is related to age and sperm progressive motility. Fertil. Steril.101, 1588–1593 (2014).

    CASArticle Google Scholar

  51. 51.

    Hamilton, M. et al. Working Party on Sperm Donation Services in the UK. Hum. Fertil.11, 147–158 (2008).

    Article Google Scholar

  52. 52.

    Dallinga, J. W. et al. Decreased human semen quality and organochlorine compounds in blood. Hum. Reprod.17, 1973–1979 (2002).

    CASArticle Google Scholar

  53. 53.

    Zhang, Y.-H., Zheng, L.-X. & Chen, B.-H. Phthalate exposure and human semen quality in Shanghai: a cross-sectional study. Biomed. Environ. Sci.19, 205–209 (2006).

    CASPubMed Google Scholar

  54. 54.

    WHO. Laboratory Manual for the examination of human semen and sperm – cervical mucus interaction. (Cambridge University Press, Cambridge, 1999).

  55. 55.

    Fernandez, J. L. et al. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil. Steril.84, 833–842 (2005).

    CASArticle Google Scholar

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Acknowledgements

We thank Karen Pooley for the management of the human donors attending the Fertility Unit at Nottingham University Hospital and for expert technical advice and assistance. We also thank Natasha White for the collection of ejaculates from a population of assistance dogs. The work was supported by University of Nottingham internal funds.

Author information

Author notes
  1. Rebecca N. Sumner

    Present address: Hartpury University, Gloucester, GL19 3BE, UK

Affiliations

  1. School of Veterinary Medicine & Science, University of Nottingham, Sutton Bonington, LE12 5RD, UK

    Rebecca N. Sumner, Gary C. W. England & Richard G. Lea

  2. Fertility Unit, East Block B floor, Nottingham University Hospital, Nottingham, NG7 2UH, UK

    Mathew Tomlinson

  3. School of Biosciences, University of Nottingham, Sutton Bonington, LE12 5RD, UK

    Jim Craigon

Contributions

R.G.L. and R.N.S. conceived of and designed the study. M.T. managed the human donors and collection of human semen samples and G.C.W.E. managed the collection of canine samples. All authors provided academic input into the paper. R.N.S. carried out the computer assisted sperm analysis, chromatin dispersion assay and subsequent analysis. J.C. provided expert statistical advice and contributed to the analysis. R.G.L. and R.N.S. wrote the paper.

Corresponding author

Correspondence to Richard G. Lea.

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Competing Interests

The authors declare no competing interests.

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Sumner, R.N., Tomlinson, M., Craigon, J. et al. Independent and combined effects of diethylhexyl phthalate and polychlorinated biphenyl 153 on sperm quality in the human and dog. Sci Rep9, 3409 (2019). https://doi.org/10.1038/s41598-019-39913-9

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Источник: https://www.nature.com/articles/s41598-019-39913-9
Call Us: (+1) 508-875-7086
Slade Veterinary HospitalSlade Veterinary Hospital

Male Breeding Soundness Exam

We offer comprehensive breeding soundness examinations for your stud dogs. This includes a complete physical examination with special attention to the testicles, penis, and prostate, as well as a complete semen evaluation. We review your male's prior breeding history, including dog sperm bank near me produced and bitches that missed. Additional testing may include baseline labwork, prostatic and testicular ultrasounds, cultures, and genetic testing.

For questions related to breeding soundness exams, please speak to one of our reproduction technicians – Linn, Kirsten, or Scott.

Semen Collection and Evaluation

The manual collection of semen is a vital component of evaluating your stud dog. The libido of your dog and the quality of the semen sample is improved by the presence of a teaser bitch. You may bring your own teaser or we can provide one for him. We separately collect the different fractions of the ejaculate and utilize a computer-assisted sperm analysis (CASA) machine for thorough evaluation. Our phase contrast microscope and CASA software is state-of-the-art and incredibly precise, providing you with the most detailed information. You will receive a detailed report of your stud dog’s semen analysis.

Your male's semen quality should be evaluated in the month prior to an expected breeding. If he is used frequently, he should be manually collected and evaluated every six months, or any time a breeding does not take.

Options for Semen

Fresh semen

We collect your male, add semen extenders to enhance the motility and lifespan of the ejaculate, and use the sample for breeding immediately with a bitch.

Fresh-chilled semen

This method of collecting semen, adding extender, slowly cooling, and packaging the specimen for shipment has made long distance breeding possible. The stud dog is manually collected at our office when the owner of the bitch notifies you that their female has reached her fertile period. With fresh-chilled semen, it is the semen, not the bitch, that does the traveling. We can arrange same-day or overnight shipment within the continental United States Monday through Saturday. We recommend that stud dog owners have their dog's semen checked for its ability to withstand the chilling process. Please see semen shipping (below) for more information.

Frozen

The purpose of freezing your dog's semen is to ensure his breeding availability. Frozen semen is used when a dog is no longer fertile, how many branches does wells fargo have, or unavailable due to a scheduling conflict. Please see semen cryopreservation (freezing) below for more information.

Semen Cryopreservation (Freezing)

Our hospital began working with International Canine Genetics in the early 1990s. We are a licensed freezing center and offer semen freezing once a month. Special arrangements can be made to mitigate scheduling conflicts. 

Consider freezing your dog's semen to:

  • Preserve his genetics for your own future breeding program
  • Provide stud service when your dog is not available (show/trial schedules, injury, illness)
  • Use as back-up for breeding involving winter shipping or difficult travel
  • Use for international breeding (Please contact us in advance for individual export regulations.)

The ideal time to collect your dog's semen for freezing is when he is between two and five years of age. The semen of older dogs may be successfully frozen, but often it is more sensitive to the extreme temperature changes in the process. We have successfully produced hundreds of puppies from frozen semen, many from semen frozen 20 or more years ago!!

How do I schedule an appointment?

Our freeze dates are typically the first Friday of every month with Dr. Gatlin. Please contact us below for further details.

  1. You may contact us at 508-875-7086 to speak with one of our reproductive technicians – Mary, Linn, Kirsten, or Scott. You may also schedule this appointment by contacting [email protected]
  2. Once your appointment is scheduled, please remember to bring the following paperwork:
  • AKC registration
  • a copy of current rabies vaccine certificate
  • a negative brucellosis test (if not done here).
  1. Please arrive 15-20 minutes in advance to complete paperwork and run a brucellosis test, if necessary.

How do you know if the semen is good enough to freeze?

Immediately after collection, the semen is fully evaluated. We tell you if the concentration and motility are sufficient to continue the freezing process. Good quality semen at the time of collection is essential to ensure good semen at the time of thaw and insemination. The sample is then extended with buffers that protect the sperm cells during the controlled temperature drop over four hours to -196 C. The semen is packaged into straws. The number of straws to be stored is determined by the initial sperm count. One frozen straw from your dog’s collection is thawed and examined for post-thaw motility and quality.

If your dog has not been collected in the last 6 to 12 months, we do recommend a dog sperm bank near me and evaluation appointment prior to the freeze date to assess the quality.

When do I know the results?

At the end of the day, we will call to inform you the number of straws stored, the post-thaw motility, and the number of breedings available from the collection. We will also email you a detailed copy of the final report.

Where is the semen stored?

After your dog’s semen is collected and frozen, it safely stored here until it is shipped to Kansas City, MO, where Zoetis Inc. maintains the largest canine semen storage bank in the world. We will help establish an account for your stud dog through Zoetis’s online portal system. You will be able to access all of your frozen semen information at www.mysecurelineage.com. Once this account is established, Zoetis will contact you regarding pricing and long-term storage information. When your semen is needed, you call them to arrange for your stored semen to be shipped to the inseminating veterinarian.

The method of collecting semen, adding extender, slowly cooling, and packaging the specimen for shipment has made long distance breeding possible. We ask that you arrange for same-day or overnight semen shipment within the continental United States Monday through Saturday. 

If your stud’s semen has not been chilled before, we do dog sperm bank near me a pre-shipment evaluation and chill check test, although this is not required. The "chill check" is done by collecting the dog's semen and putting it through the same chilling process as if it was being shipped. We evaluate its quality every 12 hours for 48 hours. This process enables us to determine if overnight shipping is adequate or if same-day service is required.

Please fill out our chilled semen shipment form so we have all the information in advance. You may also contact our office to speak with one of our reproductive dog sperm bank near me semen shipments require significant planning due to strict regulations. It is the client’s responsibility to research specific destination country regulations. Please contact a staff member to discuss further. You and our reproductive liaisons will need to work together to make certain that all of the regulatory requirements are met for international shipments.

Canine brucellosis is a highly contagious infection caused by a bacteria, Brucella canis. Infected dogs develop problems including infertility, spontaneous abortions, stillborn puppies, and systemic illness. Treatment is incredibly difficult and often results in euthanasia. This disease is also zoonotic, which means it can also be spread to people. Due to these concerns, we require negative brucellosis testing on all dogs and bitches every 3 months. This test can be run by most veterinary laboratories, although we do offer in-house testing, if necessary.

Kirsten evaluating semen.

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Источник: https://sladevet.com/index.php/reproduction/stud-dog-management

Collect and Freeze Semen

OUR FREEZING CENTER WILL BE CLOSING AT THE END OF 2021. ALL SEMEN WILL BE SENT TO ZOETIS OR NC STATE VET SCHOOL FOR LONG TERM STORARGE. 

WE ARE CURRENTLY NOT DOING FREEZES, DUE TO THE PANDEMIC. HOWEVER YOU CAN CONTACT NC STATE VET SCHOOL FOR INFORMATION ABOUT THEIR FREEZING Dog sperm bank near me CALL  (919) 513 6300 option 2 or  email to [email protected]

We are a freezing center for Zoetis. We do store semen at our facility. If you are going to be using us in the near future for your dog's reproductive needs, you can go ahead and have semen shipped to us. However please let us know that you are having semen shipped prior to shipping it to us. 


Please contact us for the Collect and Freeze requirements and process. Each collection has to be scheduled in advance, and collections are only done first thing in the mornings. The process takes about 6 hours, even though the collected dog is only in the office for about 30 minutes. 

Collect & Freeze Information: 

Q. What do I need to do to collect semen on my stud dog?

A. That's an easy one! Just call our office at the number listed and we can make you an appointment. Collections for freezing must be done at 9am, no later. It takes 6 hours to take semen thru the freezing process; however the dog is only here for about 20-30 minutes.


Q. What will I need to bring to my appointment?

A. You will need to bring the following:

  1. You are required to bring your AKC or other registration papers verifying that you are a current owner of the stud. (for unregistered dogs this does not apply)
  2. You will need a copy of your DNA Profile, or we can collect the DNA sample and send it in for an additional fee.
  3. You will need a current Brucellosis test.
  4. A semen freezing contract must be completed, since we are a freezing center for Zoetis.

Q. What if I want to send my stud with an agent for collection?

A. Please provide that person with all the items above and payment. Please note that the semen contract MUST be signed by the STUD DOG OWNER. The semen will be collected and stored in Stud owners' name.


Q. Can I let someone else collect my stud and let them own the semen?

A. Yes and No. Semen Ownership can be transferred to persons, not listed as an owner on the registration papers. The semen will need to first be registered to the stud dog owner and then transferred to the new owner.


Q. Is there a cost for annual semen storage?

A. Yes, the annual storage fees are ~$120.00 per year.


Q. How do I arrange to ship my frozen semen?

A. Frozen semen shipping should be planned ahead of time. Please remember that there are a limited number of shipping tanks available for use. It takes 24 hours to charge a shipping dewar. Extra fees do apply for STAT charging and weekend/holiday deliveries.

Источник: https://plantationanimalhospitalnc.com/collect-and-freeze-semen

Services We Offer


Male Canine Reproductive Services

If you're ready to breed your stud dog now, or are concerned about the quality of the dog's semen, or if you're just looking to preserve your dog's breeding ability long after he's gone, Innovative Canine Reproduction has solutions for you.


Collection & Evaluation

Most male dogs are easily collected in the office. Semen quality and quantity is assessed.
 

Frozen Semen, Preparation, Storing and Shipping

Frozen semen technology allows for the infinite preservation of semen. This allows a male's reproductive capacity to outlive him. Additionally, a male’s reproductive capacity can be owned by multiple breeders. It can be dispersed to multiple locations awaiting use including being shipped over international borders when shipping a dog is not practical. Innovative Canine Reproduction stores semen at one of the largest canine sperm banks in the country.

Frozen semen is shipped in dry shippers cooled by liquid nitrogen. This is a safe and effective means of transport. It is shipped overnight via Federal Express domestically. International shipment times vary.

Infertility

There are many causes of infertility dog sperm bank near me male dogs. The older a dog is and the more he is used, the greater the likelihood of infertility. One of our areas of interest is identifying and addressing the causes of infertility. Since the restoration of fertility is often difficult, storing semen early in a stud dogs life is a wise investment for any breeding program.

Female Canine Reproductive Services

When a bitch comes into season, timing is critical in every step of the breeding process -- from pinpointing ovulation through progesterone testing to selecting the timing of insemination. Innovative Canine Reproduction provides all these services so responsible breeders can maximize the dog's chances at successful pregnancy.

Breeding Timing by Tradeking binary options of the expense, and critical nature of the appropriate time of insemination, progesterone testing is critical to the breeding process. ICR provides accurate progesterone testing and can pinpoint the most optimal day(s) to begin breeding.

Successful breeding depends on determining the optimum time for insemination. Factors that need consideration include how the semen is prepared, how a particular female cycles and her reproductive history. We maintain equipment dedicated and calibrated for progesterone testing, this allows us to provide accurate same day results Monday through Saturday.

Artificial Insemination

Artificial Insemination (AI) techniques include vaginal, trans-cervical and surgical insemination. The best method for a particular breeding is determined by consultation.

Vaginal insemination is the simplest. It can be done by breeders, owners and veterinarians with success. The technique has application for neat (side by side) and for chilled semen.

Using trans-cervical insemination, the semen is deposited into the uterus through the cervical canal. Two techniques are used to accomplish this procedure. At Innovative Canine Reproduction most trans-cervical inseminations are done under sedation using the Norwegian pipette, a specially designed tool developed in Scandanavia and used widely throughout Europe. Dr. Mantell is one of a small number of people in North America trained in this technique. It is a safe and effective method of intra-uterine insemination. The second technique uses optical instruments to visualize the cervix. We use this technique when body conformation precludes use of the Norwegian pipette. The most appropriate technique for an individual is best determined by consultation.

Surgical insemination is the gold standard for artificial insemination. It is the only technique that allows direct examination of the uterus and ovaries if needed. Surgical insemination bypasses any problem related to the condition of the vagina and cervix. It requires general anesthesia and abdominal surgery. It has broad application for canine reproduction in that veterinarians are universally familiar with similar abdominal surgeries.

The most appropriate technique for an individual dog is best determined by consultation.

Whelping Management & C-sections

Care of the whelping female must be prepared for locally. We are happy to consult with your veterinarian when our expertise is needed. Arrangements to care for our local breeders are made individually.

Источник: https://www.innovativecaninereproduction.com/services.html

Animal Breeding and Reproductive Services
in Frederick County

Consistent winner of Frederick's Best Vet, 2007-2021!

Buckeystown Veterinary Hospital offers reproductive services for both companion and farm animal species.

The following reproductive services are offered for the species listed below. If you require a breeding or reproductive service and do not see it listed, contact us for a consultation at:

Breeding & Reproduction Services for Dogs

  • Pre-breeding consultation and evaluation
  • Brucellosis testing
  • Progesterone testing
  • Vaginal cytology
  • Breeding management
  • Artificial insemination
  • Surgical insemination
  • Pregnancy diagnosis by ultrasound
  • “Puppy count” X-rays
  • Dystocia management
  • Caesarian section
  • Neonatal care
  • Semen collection and evaluation
  • Semen collection and shipment
  • Semen collection, freezing, and storage

Breeding & Reproductive Services for Horses

  • Pre-breeding consultation and examination
  • Breeding soundness examination
  • Breeding management
  • Artificial insemination using fresh, chilled, or frozen semen
  • Pregnancy diagnosis by ultrasound
  • Twin management
  • Late-term mare evaluation
  • Neonatal care

Breeding & Reproductive Services for Cattle

  • Breeding management
  • Pregnancy diagnosis by palpation or blood test
  • Neonatal care

Breeding & Reproductive Services for Small Ruminants

  • Breeding management
  • Pregnancy diagnosis by ultrasound
  • Neonatal care

Breeding & Reproductive Uk phone country code from usa for Llamas & Alpacas

  • Breeding management
  • Evaluation of problem breeders
  • Pregnancy diagnosis by palpation and ultrasound
  • Neonatal care
Источник: https://www.buckeystownvet.com/veterinary-services/breeding-reproduction.html

Semen collection in the dog

local food banks northern virginia This review will discuss semen collection in the dog. Semen samples may be collected from male dogs for the purposes of artificial insemination, cryopreservation or diagnosis. The materials needed for semen collection depend on which method is used and the collector's level of expertise with this procedure. At minimum, two sterile centrifuge tubes or specimen cups can be used to collect semen as it is ejaculated (for the combined first and second fractions and for the third fraction). The most common method for semen collection in the dog is by digital stimulation. Under ideal conditions, this procedure is performed in the presence of an estrous bitch. Initially, the dog's penis is vigorously massaged through the prepuce at the level of the bulbus glandis (caudal-most aspect of the prepuce) until a partial erection develops (initial engorgement of the bulbus glandis). The prepuce is quickly retracted past the bulbus glandis and firm constant pressure is applied to the penis behind the bulbus glandis by squeezing the penis between index finger and thumb. Pelvic thrusting may occur following application of pressure behind the bulbus glandis during the development a "full" erection. The ejaculate is composed of three fractions: first (sperm-poor), second (sperm-rich) and third (prostatic fluid). In addition to digital stimulation of the penis, spermatozoa have been collected from dogs using electroejaculation and pharmacologic methods.

Источник: https://pubmed.ncbi.nlm.nih.gov/15993482/

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