Effect of different levels and sources of zinc supplementation on quantitative and qualitative semen attributes and serum testosterone level in crossbred cattle (Bos indicus x Bos taurus) bulls

An experiment was conducted on 16 crossbred bulls (about 2 years of age, 316.2+/-0.77 kg average body weight), divided into groups I, II, III and IV to study the effect of different levels of Zn supplementation from inorganic and organic sources on semen quality. The animals in the first 3 groups were supplemented with 0, 35 and 70 ppm Zn from Zn sulfate, respectively and the animals in-group IV were supplemented with 35 ppm Zn as Zn propionate. Semen collection and evaluation was done in the first month (to assess semen quality at the start of the experiment) and 7th, 8th and 9th month of experimental feeding to evaluate the effect of supplemental Zn on semen attributes. We gave 6 months for Zn feeding, so that 3 sperm cycles of spermatogenesis had passed and the collected semen reflected the complete effect of Zn supplementation. Six ejaculates from each bull were collected and evaluated for semen quantitative (ejaculate volume, sperm concentration and sperm number per ejaculate) and qualitative characteristics (semen pH, mass motility, individual motility, sperm livability percent and abnormal sperm percent, percent intact acrosome, bovine cervical mucus penetration test, hypo-osmotic sperm swelling test) and activity of seminal plasma enzymes i.e., alkaline phosphatase, acid phosphatase, GOT and GPT. Testosterone level in the blood serum of crossbred bulls was also estimated. Mean values of semen quantitative and qualitative characteristics at the start of the experiment were statistically non significant (P > 0.05) in all the crossbred cattle bulls, however, there were statistically significant differences among the bulls of different groups after 6 months of zinc supplementation. Mean ejaculate volume (mL) was 2.37, 4.70, 5.86 and 6.38, respectively in groups I to IV, indicating a statistically significant (P < 0.05) higher semen volume in Zn-supplemented groups as compared to the control group of bulls. Similarly, sperm concentration (million.mL(-1)), live sperm (%) and motility (%) were significantly (P < 0.01) higher in Zn-supplemented groups as compared to the control group. The results of BCMPT and HOSST revealed a significant improvement in sperm functional ability in all the groups supplemented with Zn as compared to the control group. The activity of alkaline and acid phosphatase in seminal plasma was significantly (P < 0.05) higher in the Zn-supplemented groups, whereas GOT and GPT activities in seminal plasma were significantly (P < 0.05) lower in the Zn propionate supplemented group as compared to the control group. Testosterone concentration (ng.mL(-1)) in blood serum was significantly higher in animals of groups III and IV, as compared to control group. It may be concluded that Zn supplementation either in the inorganic or organic form in the diet of crossbred bulls improved the qualitative and quantitative attributes of semen; however, the number of sperm per ejaculate, mass motility and semen fertility test like bovine cervical mucus penetration was significantly higher in bulls given Zn in an organic form (Zn propionate) as compared to an inorganic form (Zn sulfate).     

Effect of prostaglandin F2 alpha on libido, seminal quantity and quality of buffalo bulls

Four mature Murrah buffalo bulls on a regular semen collection schedule of twice a day, two days per week, were injected with 30 mg prostaglandin F2 alpha THAM salt(PGF) intramuscularly 30 minutes prior to each alternate first ejaculation for six weeks. Effects of PGF on time to initial false mount on the decoy, time to ejaculation after two false mounts (reaction time), seminal volume, sperm concentration, total sperm output, motility of fresh and thawed semen and the semen doses obtained per collection were evaluated. Treatment caused significant (P < 0.01) reduction in the time to first false mount and the reaction time for the first ejaculations but had no effect (P > 0.05) on these for the second ejaculations. Values for other quantitative and qualitative parameters did not differ (P > 0.05) due to PGF treatment. It is concluded that PGF at the dosage and frequency of administration used may be of some value in improving libido in low-libido bulls but does not alter the reproductive capacity of buffalo bulls.     

Advances in cryopreservation of stallion semen in modified INRA82.

In the procedure used in this paper, semen was first diluted in INRA82+2% egg yolk (E1) at 37 degrees C. Before or after cooling to 4 degrees C, semen was centrifuged and diluted in E1+2.5% glycerol (E2). Cooled semen was frozen in 0.5-ml straws. Straws were thawed at 37 degrees C for 30s. For fertility trials, frozen ejaculates were used only if total post-thaw motility was above 35%. Most mares were inseminated two times before ovulation with 400 x 10(6) total spermatozoa every 24h. This paper presents post-thaw motility (CASA) and fertility results obtained when some steps of the procedure were evaluated.Use of the first three jets of ejaculate before the centrifugation did not improve post-thaw motility compared to use of the whole semen (25% versus 25%, 2 stallions x 12 ejaculates, P>0.80). When the first dilution was performed in E2 at 22 degrees C instead of in E1 at 37 degrees C, motility was slightly improved (38% versus 36%, n>283 ejaculates per group, P<0.04) but fertility was similar (51% versus 58%, n>196 cycles per group, P>0.10). Coating the spermatozoa with 0.5, 1, 2, 4 and 8mM of Concanavalin A resulted in unchanged post-thaw motility (6 stallions x 3 ejaculates, P>0.05). The extender E2 was modified or supplemented with different substances. Increasing egg yolk concentration from 2 to 4% (v/v) did not increase post-thaw motility (42% versus 34%, 6 stallions x 2 ejaculates, P>0.05). Different glycerol concentrations (range: 1.7-3.7%) had no significant effect on post-thaw motility even though 2.4-2.8% resulted in a nonsignificant higher motility (7 stallions x 2 ejaculates, P>0.05). Glutamine at 50mM in E2 improved post-thaw motility compared with no glutamine (49% versus 46%, n>584 ejaculates per group, P<0.0001) but not fertility (53% versus 54%, n>451 cycles per group, P>0.80). Thawing at 75 degrees C for 10s slightly increased motility after 120 min at 37 degrees C (6 stallions x 1 ejaculate, P<0.05) but no effect on per-cycle fertility was noted (32% (19 cycles) versus 41% (17 cycles), P>0.50). When post-thaw dilution was performed using a fixed molarity multi-step system (25 mOsm per step) from various osmolarities (900-690 mOsm) to 365 mOsm, motility was unaffected compared with dilution in one step (36% versus 38%, 6 stallions x 1 ejaculate, P>0.20).     

Epididymal sperm from the African buffalo (Syncerus caffer) can be frozen successfully with AndroMed and with Triladyl but the addition of bovine seminal plasma is detrimental

Numerous diseases are carried and can be transmitted from the African buffalo (Syncerus caffer) to livestock. Buffaloes free of specific diseases (BFSD) are thus in demand amongst game farmers. Current BFSD derive from a small genetic pool and hence there is a special interest in bringing new genetic material into such herds. In this study epididymal sperm from 16 mature African buffalo bulls was frozen with Triladyl and AndroMed extender (Minitüb, Tiefenbach, Germany) with and without addition of bovine seminal plasma. Post-thaw motility, longevity and acrosomal integrity were compared. In all but one animal, post-thaw motility was higher, although not always significant, if sperm was frozen with Triladyl than with AndroMed. Seminal plasma was detrimental to the post-thaw motility. Neither semen extender nor seminal plasma had an influence on post-thaw acrosomal integrity. It can be concluded that bovine seminal plasma at a concentration of 10% is detrimental rather than beneficial for the post-thaw motility of African buffalo sperm. Even though being inferior AndroMed does, however, have the advantage that it is a defined semen extender and therefore clearly has a lower risk of contamination.     

Dialysis of boar semen prior to freezing-thawing: its effects on post-thaw sperm characteristics

Low-molecular weight components of the seminal plasma have a detrimental effect on sperm function. The present study was undertaken to evaluate the effect of the removal of low-molecular weight components by dialysis on sperm characteristics prior to and after freezing. Semen, collected from 5 boars, was extended in Kortowo-3 extender (K-3, Poland) and cooled for 3h (control non-dialysis) or dialyzed for 5h in semi-permeable dialysis bags of 12-14kDa molecular weight cut-off prior to freezing. The semen samples were diluted in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or lactose-lyophilized lipoprotein fractions-glycerol extender (lactose-LPFo-G), packaged into aluminum tubes and frozen in a controlled programmable freezer. Pre-frozen and frozen-thawed spermatozoa were evaluated for motility, plasma membrane (SYBR-14 and propidium iodide) and acrosome integrity, mitochondrial function (Rhodamine 123) and ATP content. The results of the study showed that dialysis significantly improved the sperm characteristics prior to freezing. Dialysis enhanced (P<0.05) post-thaw sperm motility, plasma membrane integrity and mitochondrial function, but had no significant effect (P>0.05) on recovery of spermatozoa with intact acrosomes. Furthermore, dialyzed spermatozoa exhibited higher (P<0.05) ATP content compared with the control after freezing-thawing. Consistent inter-boar variability was detected mainly in dialyzed semen following freezing-thawing. These results indicated that the improvement in sperm quality characteristics prior to freezing and the post-thaw sperm recovery were due to the removal of low-molecular weight components from the seminal plasma. It can be suggested that dialysis is effective in improving the post-thaw quality of boar spermatozoa and has also great practical importance in improving the protocols for cryopreservation of semen. Dialysis may also contribute to a better understanding of different mechanisms underlying cryo-induced damage to boar spermatozoa.     

Factors affecting the removal of low-molecular-weight fractions (LMWF) from egg yolk and seminal plasma in extended semen by dialysis: effect on post-thaw sperm survival

Three factors affecting dialysis of bovine semen were studied. These factors were (1) dialysis rates of egg yolk, seminal plasma, and glycerol, (2) temperature (37 degrees C, 5 degrees C, and while cooling from 37 to 5 degrees C), and (3) dialysis ratios between retentate and dialysate (1:1, 1:10, 1:20, 1:50, and 1:100). Ninety percent of the low-molecular-weight fraction (LMWF) from seminal plasma, egg yolk, and glycerol was removed from the retentate in a 2-hr period at 5 degrees C, and only slight changes were detected after the third hour of dialysis. Temperature affected dialysis and was faster at 37 degrees C. It was also found that a 1:20 dialysis ratio was sufficient to obtain 90% clearance of the LMWF. The effect of sperm dilution ratio, dialysis ratio, and exchange of the LMWF from egg yolk and/or seminal plasma for buffer systems was also studied. An improvement in post-thaw motility of spermatozoa (P less than 0.05) was obtained when the LMWF from both seminal plasma and egg yolk were replaced. A third experiment was conducted to study the effect of different combinations between the buffer systems, TEST and Na citrate, in the dialysate. The results indicated that a 1:1 combination of iso-osmotic solutions (320-325 mOsm/Kg, pH 7.0) between these two buffers, with 5% glycerol (v/v), yielded significant (P less than 0.05) sperm post-thaw motility as compared with the individual use of TEST-glycerol or Na citrate-glycerol. Dialyzed samples also yielded sperm post-thaw motility higher than that of the nondialyzed samples. Colloidal materials in the dialysate did not affect survival of spermatozoa.     

Effect of age and environmental factors on semen quality, glutathione peroxidase activity and oxidative parameters in simmental bulls

Taking into account that semen quality depends on animal age and climate conditions and that oxidative stress has been reported to be a common cause of infertility, the objective of this study was to monitor indicators of oxidative stress and antioxidant protection during four seasonal periods in service bulls of various age to get better insight into the significance of these factors upon evaluating service bull semen. The research was conducted over a year on 19 Simmental service bulls. Animals were divided into two groups according to age; Group I consisted of younger bulls aged two to four yrs (n = 9), and Group II was comprised of older bulls aged five to ten yrs (n = 10). Semen samples were obtained once in the middle of every seasonal period and blood samples for biochemical analysis were collected by jugular venipuncture immediately after ejaculate collection. The activity of total glutathione peroxidase (T-GSH-Px), selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase (non-Se-GSH-Px), together with the intensity of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) and oxidative protein damage (protein carbonyl content (PCC)) were measured in seminal plasma. In samples of spermatozoa and blood serum, the activity of Se-GSH-Px and TBARS and PCC concentrations were determined. Older service bulls had significantly higher ejaculate volume in summer in comparison with younger bulls, whereas the number of spermatozoa and progressive motility percentage did not significantly vary with age. Younger animals had lower progressive motility percentage during summer than in spring, with more intensive oxidative processes observed in seminal plasma (TBARS) and spermatozoa (TBARS and PCC). Based on the results presented here, it can be concluded that younger bulls are more sensitive to elevated ambient temperatures during the summer, when intensified prooxidative processes in semen plasma and spermatozoa eventually led to decreased sperm progressive motility with consequential semen quality deterioration.     

Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation

Effects of seminal plasma on post-thaw motility and membrane integrity of cryopreserved horse spermatozoa were investigated. Carboxyfluorescein diacetate staining was used for the assessment of sperm membrane integrity. Adding 30% of seminal plasma from stallions with high post-thaw sperm motility to ejaculates from stallions with low post-thaw sperm motility increased progressive motility from 24.0 +/- 1.6 to 34.5 +/- 1.9% (P < 0.05) and membrane integrity from 27.0 +/- 2.1 to 34.3 +/- 2.3% membrane-intact spermatozoa (P < 0.05). Conversely, the addition of seminal plasma from stallions with low post-thaw sperm motility to ejaculates from stallions with high post-thaw motility decreased progressive motility from 36.0 +/- 1.6 to 30.0 +/- 2.7% (P < 0.05) but did not induce changes in membrane integrity. Seminal plasma from stallions with opposite post-thaw motility therefore clearly influenced the resistance of spermatozoa to the freezing and thawing process. We conclude that the individual composition of seminal plasma affects the suitability of stallions for semen cryopreservation.     

Seminal plasma damages sperm during cryopreservation, but its presence during thawing improves semen quality and conception rates in boars with poor post-thaw semen quality

To determine the effects of seminal plasma during and after cyopreservation on post-thaw sperm functions in semen from poor freezability boars, seminal plasma was removed immediately after collection, and sperm was subjected to cooling and freezing. Removal of seminal plasma did not significantly affect post-thaw sperm motility in good freezability boars; however, in boars with poor freezability, it increased post-thaw motility relative to control sperm cooled with seminal plasma (64.5+/-3.4% vs. 30.9+/-3.1%, P<0.01). Freezing sperm without seminal plasma increased both loss of the acrosome cap (37.5+/-1.6% vs. 18.4+/-2.8%, P<0.01) and expression of a 15 kDa tyrosine-phosphorylated protein (capacitation marker) in thawed sperm relative to controls; the addition of 10% (v/v) seminal plasma to the thawing solution significantly suppressed both changes and increased conception rate to AI (70% vs. 9% in the control group, P<0.05). In conclusion, our novel cryopreservation and thawing method increased the success of AI with frozen-thawed porcine semen, particularly from boars with poor post-thaw semen quality.     

Six novel single-nucleotide polymorphisms in SPAG11 gene and their association with sperm quality traits in Chinese Holstein bulls

Sperm-associated antigen 11 (SPAG11) is predominant in the male reproductive tract. Similar to β-defensin, aside from its antibacterial activity, SPAG11 also has an important role in male reproductive function. In the present study, the association of bovine SPAG11 gene polymorphism with sperm quality traits was examined, including ejaculate volume, sperm concentration, fresh sperm motility, post-thaw cryopreserved sperm motility, and deformity rate of bull semen. Six novel single nucleotide polymorphisms (SNPs) of the SPGA11 gene were investigated in 426 normal mature Chinese Holstein bulls using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), PCR-restriction fragment length polymorphism (PCR-RFLP), created restriction site-PCR (CRS-PCR), and DNA sequencing methods. Linkage disequilibrium analysis showed that g.1306G>A and g.1454G>A (SNP-1), and g.16904G>T, g.16974C>T, and g.17000A>G (SNP-2) are completely linked, respectively. Correlation analysis showed the SNP-2 marker had a marked effect on fresh sperm motility and sperm concentration (P<0.05). SNP-3 g.22696T>C had a marked effect on post-thaw cryopreserved sperm motility (P<0.05) and deformity rate (P<0.01). However, the presence of SNP-1 was not correlated with the sperm production traits (P>0.05). Furthermore, association analyses of the 8 haplotypes constructed from the 17 combined haplotypes and reproductive traits showed that the bulls with the combined haplotype H5H6 (GGT/TTC) have the highest ejaculate volumes and the bulls with combined haplotypes H1H1 (AAT/TTT) and H1H6 (AGT/TTC) had the highest fresh and post-thaw sperm motilities, respectively. These results indicate that new molecular markers associated with sperm quality traits can be used in marker-assisted selection in bull breeding programs.     

Comparison of seminal quality in Holstein bulls as yearlings and as mature sires

Semen quality was compared in 5 Holstein bulls from samples collected as young sires (yearlings) and again as mature bulls after a mean interval of 1,265 d. At both sampling periods, the semen was examined for ejaculate volume, sperm numbers, post-thaw progressive motility and sperm viability. Sperm viability was assessed on cryopreserved samples with fluorescent SYBR-14 to stain living spermatozoa and propidium iodide (PI) to identify dead spermatozoa. The fluorescent populations of stained spermatozoa were quantified by flow cytometry. The percentages of living spermatozoa for the individual bulls, as determined by green fluorescence of SYBR-14, ranged from 44 +/- 3.1 to 54 +/- 0.3 for yearlings, and from 38 +/- 1.5 to 55 +/- 1.0 for mature sires. No differences in sperm viability were found between samples taken from yearling bulls and those of mature bulls. The percentage of spermatozoa stained with SYBR-14 was negatively correlated (r = -0.97; P = 0.0001) with the percentage of dead spermatozoa as indicated by PI staining. Comparisons of identical samples run on 2 different flow cytometers indicated that either flow instrument could be used to assess sperm viability. Although the individual bulls differed (P < 0.05) in ejaculate volume and sperm numbers as yearlings, they did not differ in these parameters as mature bulls. The average number of spermatozoa per ejaculate changed as a result of maturation, increasing from 6.2 +/- 1.0 to 10.7 +/- 1.1 x 10(9). Aging was significantly correlated with ejaculate volume (r = 0.76; P = 0.01) but not with the total number of spermatozoa per ejaculate (r = 0.51; P = 0.13). The maturational changes that occurred in the 5 bulls were minimal with the exception of the increased volume of the ejaculate and the number of spermatozoa per ejaculate.     

Dialysis of bovine semen and its effect on fresh and freeze-thawed spermatozoa

The effect of the removal of the low-molecular-weight fraction (LMWF, less than 12,000-14,000 Da) from the seminal plasma present in extended semen by dialysis and by centrifugation (1,376g for 20 min at 5 degrees C) were compared with the current methods of freezing bovine semen. Significantly higher sperm post-thaw motility (P less than 0.05) was obtained in the dialyzed samples than with the other two methods. The appropriate time and temperature for dialysis of semen was also studied. Semen aliquots were dialyzed (1:50, retentate:dialysate) for 30 min, 1 or 2 hr at 5 degrees C, and during the cooling process from 37 to 5 degrees C over a 2-hr period. Superior sperm motility (P less than 0.05) in prefreeze and post-thawed samples was observed when semen was dialyzed for 1 or 2 hr during the cooling process as compared with that of semen dialyzed at 5 degrees C. A third experiment was conducted to establish the effect of the use of dialysis bags of different molecular weight cutoffs (MWCO) on sperm motility. Semen samples were dialyzed (1:50) during the cooling process in dialysis bags of 1,000, 3,500, 6,000-8,000, 12,000-14,000, 25,000, and 50,000 MWCO. No statistical differences (P greater than 0.05) in sperm post-thaw motility were found after evaluation of the number of cells that passed through the Sephadex filter and all the dialyzed values obtained were significantly (P less than 0.05) superior to the results obtained with no dialysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prefreezing and post-thaw semen characteristics of five ram breeds collected by electroejaculation

Rams representing five breeds were electroejaculated twice weekly, during a three-week collection period. Ejaculates were evaluated for volume and concentration before freezing and for rate of motility and percentages of motile and abnormal cells both before and after freezing. Interactions between breed and collection period were evident (P<0.05) for semen volume and post-thaw values for rate of motility and percentage motile cells. Breeds differed (P<0.05) in these traits during some periods. In contrast, pre-freezing observations of rate of motility, percentage motile and abnormal cells and post-thaw percentage abnormal cells did not differ (P>0.15) among breeds. Sperm concentration per ejaculate tended to vary (P=0.11) among breeds. Semen characteristics frequently varied across collection periods. Rams within a breed differed (P<0.01) in all semen traits except post-thaw rate of motility and percentage motile cells. Semen was negatively affected by the freezing and thawing procedure. Ram within a breed and ejaculate within ram should be considered when selecting electroejaculated semen for freezing and subsequent use in artificial insemination.     

Evaluation of relative fertility of cryopreserved goat sperm

This study was designed to compare differences in the in vivo fertility of cryopreserved goat semen assessed by heterospermic insemination with differences in in vitro analyses. Five groups of does were inseminated with mixed frozen-thawed semen from different pairs of bucks. The percentage of offspring sired by each buck in the pair was compared with the relative ability of spermatozoa from that frozen-thawed ejaculate to penetrate zona-free hamster ova, relative post-thaw acrosomal integrity, ability to undergo an acrosome reaction during in vitro capacitation, and assessments of sperm motility. In 4 of the 5 different insemination pairs, the ratio of offspring born was other than 1:1. Acrosomal integrity, ability of spermatozoa to undergo an acrosome reaction, and parameters of sperm motility were not correlated with differences in relative fertility in this experiment using ejaculates from fertile bucks. The ability of spermatozoa to fuse with the oocyte plasma membrane was highly correlated with relative in vivo fertility (R(2) = 0.78, P = 0.04). This suggests that fusion with the oocyte plasma membrane is an event in the fertilization process in which significant variation exists among fertile bucks. Assessment of ability of spermatozoa to fuse with zona-free hamster ova may contribute to analysis of post-thaw fertility of frozen-thawed buck semen.     

Age-related differences of semen quality,seminal plasma,and spermatozoa antioxidative and oxidative stress variables in bulls during cold and warm periods of the year

The aims of this study were to determine the presence and quantities of antioxidative status and oxidative stress (OS) variables in the seminal plasma and spermatozoa of bulls of varying age during cold and warm periods of the year, and to establish the correlation of these variables with semen quality parameters. The study was conducted on two groups each comprising nine Simmental bulls: one group contained younger animals (aged 2 to 4 years) and the second older animals (aged 5 to 10 years). Semen samples were collected using an artificial vagina for biochemical analysis. Seminal plasma and spermatozoa activities of total superoxide dismutase (TSOD), manganese superoxide dismutase (MnSOD), copper–zinc superoxide dismutase (CuZnSOD), catalase (CAT), selenium-dependent glutathione peroxidase, reduced glutathione and concentrations of total protein (TP), thiobarbituric acid reactive substances (TBARS) and protein carbonyl content (PCC) were determined. Several antioxidants in seminal plasma were also determined: total glutathione peroxidase (TGSH-Px), selenium-independent glutathione peroxidase (Non-SeGSH-Px), uric acid, albumins (ALB) and alkaline phosphatase (ALP). Significantly higher spermatozoa motility was observed during the cold v. warm period, and a significantly higher volume and total number of spermatozoa per ejaculate was observed in older than in younger bulls. Significantly higher values of ALP, TP and ALB were found in seminal plasma of older bulls than in younger bulls during the warm period. The seminal plasma of younger bulls showed significantly higher activities of TSOD, MnSOD, CuZnSOD, TGSH-Px and Non-SeGSH-Px. Younger bulls had significantly higher PCC concentration and activity of CAT in seminal plasma than older bulls during the cold period. Significantly higher concentrations of PCC and TBARS, and activities of TSOD, MnSOD and CuZnSOD were established in spermatozoa of the younger than in older bulls during the warm period. It could be concluded that antioxidative and OS variables differ significantly depending on bull age and time of year. Younger bulls were more sensitive to elevated ambient temperatures during the warm period, when the higher enzymatic antioxidative protection in seminal plasma and spermatozoa were insufficient to counteract the intensive oxidative processes in spermatozoa, which eventually resulted in decreased spermatozoa motility. The estimation of antioxidative and OS variables in seminal plasma and spermatozoa may have practical value for the assessment of bull semen quality.     

Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions

The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejaculate volume (r = −0.43, P = 0.05) and total IGF1 content (ng) per ejaculate (r = −0.58, P = 0.006), whereas overall pregnancy rate was positively related to sperm concentration (r = 0.56, P = 0.01). The abundance of three proteins known to be involved in carbohydrate metabolism in sperm was positively related to fertility. Furthermore, the abundance of four seminal plasma proteins were identified as being negatively related to fertility; these were identified as kallikrein-1E2 (KLK2), clusterin, and seminal plasma proteins 1 (SP1) and 2 (SP2). Abundance of cysteine-rich secretory protein 3 (CRISP3) was positively related to first cycle conception rate (r = 0.495, P = 0.027) and may provide a good marker of fertility. Based on stepwise regression analysis, clusterin and SP1 in seminal plasma together with sperm citrate synthase were predictive of fertility (r = 0.77, P < 0.0001). This study identified proteins within sperm and seminal plasma that could serve as biomarkers of semen quality and fertility in stallions.     

Seminal plasma non-heparin binding proteins (NHBP) reduce the cryoinjury to buffalo cauda epididymal spermatozoa induced by heparin binding proteins (HBP)

Previous cryopreservation studies with buffalo cauda epididymal spermatozoa have reported a deleterious effect of seminal plasma heparin binding protein (HBP). The amount of HBP used in these studies was meager compared to the normal level of HBP in the buffalo ejaculate, still the damage induced upon the spermatozoa was substantial when compared to that incurred to the spermatozoa during routine freezing of ejaculated semen. Thus there might be some factor(s) in the seminal plasma, which reduce the deleterious effect of HBP on spermatozoa during cryopreservation of ejaculated semen. This study was conducted to investigate for the presence of any such factor in buffalo seminal plasma. Seminal plasma proteins were separated on their heparin binding properties as heparin binding (HBP) and non-heparin binding (NHBP). The separated proteins were added to the extender of buffalo cauda epididymal semen for cryopreservation either alone or in combination. The spermatozoa were assessed for progressive motility, viability, acrosomal integrity and response to hypo-osmotic solution test (HOST) at prefreeze and post-thaw stages of cryopreservation. NHBP was found to provide some degree of protection to buffalo spermatozoa against cryopreservation stress as well as the deleterious effect of HBP during cryopreservation.     

Estrogens and prostaglandin F2alpha in the semen and blood plasma of stallions

A study was performed to determine the levels of estrogens and prostaglandin F(2)alpha in the stallion ejaculate. Simultaneous semen and blood plasma samples were collected from 19 stallions, 2 weeks apart, during the breeding season. Although not statistically different, the total mean estrogen content tended to be higher in seminal plasma (4447 pg/ml) than in blood (2497 pg/ml). A tendency was found for higher mean estrone sulphate concentrations than for total free steroid in both seminal (4116.1 vs 330.5 pg/ml) and blood plasma (2447.1 vs 49.5 pm/ml). Mean concentrations of estrone in ejaculate and blood plasma were 257.1 +/- 267.0 (SD) and 9.5 +/- 5.4 pg/ml, respectively. Estradiol-17beta concentrations were 73.4 +/- 87.4 and 40.0 +/- 27.6 pg/ml in ejaculates and blood plasma, respectively. Mean PGF(2)alpha concentrations tended to be much higher than total estrogens (1106.8 +/- 1636.4, SD, vs approximately 260 ng/ejaculate, respectively). To our knowledge this is the first report of PGF(2)alpha and estrogen concentrations in the stallion ejaculate.     

Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing-thawing

Whole ejaculate or sperm-rich fraction, collected from four sexually mature boars, was frozen in an extender containing lactose-hen egg yolk with glycerol (lactose-HEY-G) or extender containing lactose, lyophilized lipoprotein fractions isolated from ostrich egg yolk and glycerol (lactose-LPFo-G), and Orvus Es Paste, respectively. The sperm samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Sperm DNA integrity was assessed using a modified neutral comet assay. Sperm characteristics such as motility, plasma membrane integrity (SYBR-14/PI), mitochondrial function (rhodamine 123) and acrosome integrity were monitored. Freezing-thawing caused a significant increase (P<0.05) in sperm DNA fragmentation, irrespective of the procedures of ejaculate collection and extender type. Sperm DNA fragmentation was significantly lower (P<0.05) in the whole ejaculate compared with the sperm-rich fraction, indicating that spermatozoa maintained in the whole seminal plasma prior to its removal for freezing-thawing procedure were less vulnerable to cryo-induced DNA fragmentation. Furthermore, spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender exhibited lower (P<0.05) DNA fragmentation than those frozen in the absence of cryoprotective substances. The levels of sperm DNA damage, as expressed by comet tail length and tail moment values, were significantly higher (P<0.05) in sperm samples frozen in the absence of cryoprotective substances. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. It can be suggested that evaluation of DNA integrity, coupled with different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, may aid in determining the quality of frozen-thawed boar semen.     

Influence of vedaprofen (Quadrisol) on quality and freezability of stallion semen

The objective of this study was to evaluate the effect of the non-steroidal anti-inflammatory drug (NSAID) vedaprofen (Quadrisol) on quality and freezability of stallion semen. Experiments were performed using 22 Franches Montagnes stallions from the National Stud in Avenches (Switzerland) randomly divided into a control and test group. Vedaprofen was given orally to all stallions of the test group at the recommended therapeutic dose (initial dose of 2mg/kg followed by 1mg/kg body weight every 12h) for 14 days. Control animals received the same amount of carrier substance. During treatment, blood samples of five stallions in both test and control group were collected for PGF(2 alpha)-metabolite (PG-metabolite) determination. Ejaculates from all stallions were collected and cryopreserved weekly for 14 weeks from September to December. Concentrations of PG-metabolite, PGF and PGE were measured in the seminal plasma of ejaculates collected 2 weeks before, during and 2 weeks after treatment. In fresh semen the volume, concentration, motility and number of normal sperm and sperm with major defects (acrosome defects, abnormal heads, nuclear vacuoles, proximal droplets, midpiece defects) were evaluated. In frozen-thawed semen samples motility as well as viability (SYBR-14/PI) were tested and the hypoosmotic swelling test (HOS) was performed. Results demonstrate that vedaprofen had no effect on blood plasma concentration of PG-metabolite but significantly inhibited both, PGF and PGE concentrations in seminal plasma. Furthermore, all quality parameters in fresh and frozen-thawed semen were not affected by vedaprofen treatment but the time of semen collection had a significant (P<0.05) effect on motility, normal sperm and sperm with nuclear vacuoles in fresh semen.