In vitro fertility evaluation of cryopreserved ram semen and its correlation with relative in vivo fertility

The present study was designed to evaluate zona-free hamster ova assay conditions for cryopreserved ram semen and to investigate the correlation between ability to penetrate zona-free hamster ova and in vivo fertility. In vivo fertility was estimated for cryopreserved semen from 5 Merino rams using heterospermic insemination. Equal numbers of postthaw motile spermatozoa from a Merino ejaculate and pooled Suffolk ejaculates were mixed prior to insemination. Each Merino ejaculate was paired with the same pool of cryopreserved Suffolk semen. Relative in vivo fertility for each Merino ram was calculated as the proportion of offspring that were sired by the Merino (range 42 to 100%). These ejaculates also differed in their ability to penetrate zona-free-hamster ova (3.6 to 9.0 penetrated spermatozoa per ovum). Differences in penetration rate were correlated with in vivo fertility (P < 0.002, R2 = 0.69). Results of these studies suggest that the zona-free hamster ova bioassay may be a useful test in the assessment of cryopreserved ram sperm fertility.     

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.     

Proportion of males with lower fertility spermatozoa estimated from heterospermic insemination

This study was designed to evaluate the proportion of males with spermatozoa detectably less fertile than the spermatozoa from other males. Previously unpublished and published data from heterospermic trials involving insemination with equal numbers of spermatozoa and resulting in at least 11 offspring from each pair of males were analyzed. The proportion of pairs in which the males sired equivalent numbers of offspring were 0.42, 0.18, 0.33 and 0.09 for trials with fresh boar semen, liquid-stored boar semen, frozen bull semen and fresh rabbit semen, respectively. The calculated proportion of males with less fertile spermatozoa were 0.36, 0.57, 0.42 and 0.70, respectively. Although these differences in fertility would not be apparent in some management systems, a high proportion of ejaculates had spermatozoa that were detectably less fertile.     

The importance of porcine sperm parameters on fertility in vivo

It would be desirable to use semen parameters to predict the in vivo fertilizing capacity of a particular ejaculate. In animal production, an ejaculate is divided into multiple doses for artificial insemination (AI); therefore, it would be economically beneficial to know the functional quality (i.e., fertility) of the semen before it is inseminated. To identify a predictive assay of the fertilizing capacity of a porcine ejaculate, we performed 4 rapid assays of sperm quality (motility, viability, physiological status as assessed by chlortetracycline fluorescence, and ATP content) on samples from 9 ejaculates, before and after a thermal stress test (42.5 degrees C, 45 min). These parameters were subsequently correlated with in vivo fertility resulting from AI with 2 sperm doses, 3 x 10(9) or 0.3 x 10(9) motile cells in 70 mL (optimal or suboptimal sperm number per insemination, respectively) from these same ejaculates. No parameter was correlated to the fertility rates obtained after inseminating with the optimal semen doses, either before or after the thermal stress test (P > 0.05). However, with respect to the animals inseminated with the suboptimal semen dose, sperm motility (the percentage of motile spermatozoa as assessed visually by microscopy) prior to thermal stress was well-correlated to fertility rates (r = 0.783, P = 0.01). The percentage of spermatozoa displaying the chlortetracycline Pattern AR (acrosome reaction) was also statistically related to fertility (r = 0.05, P = 0.04), but the biological importance of this relationship is questionable given the small variation among ejaculates (range: 0 to 2%). No other sperm parameter was significantly related to fertility rates in this group (P > 0.05). These data, therefore, indicate that sperm motility is a useful indicator of sperm fertilizing capacity in vivo. Moreover, to identify a predictor of semen fertility it is critical that the number of spermatozoa used during insemination is sufficiently low to detect differences in sperm fertilizing efficiency.     

Identification of embryo paternity using polymorphic DNA markers to assess fertilizing capacity of spermatozoa after heterospermic insemination in boars

Differences in sperm fertilizing capacity of males often remain undetected by routine semen parameters. Heterospermic insemination with equal numbers of spermatozoa from 2 males is an accurate method for assessing differences in fertility. Use of heterospermic insemination depends on a reliable, efficient assay to identify paternity of conceptuses or offspring. In this study, polymorphic DNA markers amplified by PCR were tested to determine paternity of Day 5 to 6 embryos. The fertilizing capacity of 2 boars (A and B) with similar semen parameters was compared after homospermic (n=14 gilts) and heterospermic (n=11 gilts) insemination. Single AI's were performed under suboptimal conditions using 1 x 10(9) spermatozoa at 12 to 24 h before ovulation to prompt differences in fertilization and to stimulate sperm competition. The fertilization rate and the number of accessory spermatozoa were determined in Day 5 to 6 embryos. Using 5 different polymorphic DNA markers, paternity could be determined in 95.8% of the embryos. Boar B sired significantly (P<0.05) more offspring than Boar A after insemination with pooled semen, and this was reflected by a significantly (P<0.05) higher number of accessory spermatozoa following homospermic insemination with semen from Boar B, although fertilization rates did not differ between the 2 boars after homospermic insemination. The results suggest that the viability of spermatozoa in the female reproductive tract contributes to differences in fertility rates of males with similar in vitro sperm quality parameters. The number of accessory spermatozoa is a more sensitive measure of boar fertility than the fertilization rate. Polymorphic DNA markers are suitable for verification of parentage even at a very early stage of embryonic development.     

Influence of sperm number and seminal plasma on fertility of progestagen-treated sheep in confinement

Progestagen-impregnated vaginal sponges + PMSG were used to synchronize oestrus in crossbred adult ewes which were inseminated 56 h after sponge removal with 0.5 ml diluted semen containing 400, 200, 100, 50 or 25 x 10(6) spermatozoa per insemination. The diluent was skim milk-citrate or pooled seminal plasma. There was no difference in reproductive performance due to the insemination medium. Fertility (no. of ewes lambing) after insemination of 400 or 200 x 10(6) spermatozoa was 68% and was similar to that observed after natural service at progestagen-induced oestrus. When less than or equal to 100 x 10(6) spermatozoa were inseminated, fertility fell markedly and the number of lambs per ewe inseminated decreased. A decrease in litter size also occurred. The data indicate that insemination of 200 x 10(6) spermatozoa, i.e. less than 10% of the number in a single ram ejaculate, allows normal conception rates in progestagen-treated ewes.     

Study of fertilising capacity of spermatozoa after heterospermic insemination in rabbit using DNA markers

The aim of this study was to evaluate the fertilising capacity of males belonging to a rabbit line selected for growth rate using heterospermic insemination and genetic markers. Semen from five males was used to make pools of three of them, and to perform homospermic insemination. Insemination was carried out in receptive multiparous lactating does with 6 million spermatozoa per insemination dose. DNA from 360 young rabbits born from heterospermic insemination, 5 sires and 42 does were amplified to nine microsatellite loci for determination of the offspring rate per male. Although each female was inseminated with the same number of spermatozoa from each male (2 million from a total dose of 6 million), sperm from one male was always dominant, notable differences being observed in the offspring among the males with similar semen quality (83-68% from dominant male versus 31-0% from non-dominant, P<0.05 ).     

Embryo production from superovulated sheep inseminated with sex-sorted ram spermatozoa

An experiment was undertaken to assess the fertilizing capacity of sex-sorted, frozen-thawed ram spermatozoa, artificially inseminated into superovulated ewes, and the quality and survivability of the resultant pre-sexed embryos. Synchronized (intravaginal progestagen pessary and GnRH) donors were superovulated using PMSG and repeat ovarian stimulation with FSH before insemination. Ewes (n=67) were inseminated with either 30x10(6) or 15x10(6) motile non-sorted (control) or 15x10(6) motile sex-sorted (sorted) frozen-thawed spermatozoa (control: C30 or C15; sorted: S15, respectively) and the resultant embryos transferred immediately into synchronized recipients (n=160). The percentage of transferable embryos, pregnancy rate and embryo survival were similar (P>0.05) across all treatments. Oocyte cleavage rate was higher for ewes inseminated with S15 (172/230; 74.8%; P<0.05) than for C15 (97/151; 64.2%) or C30 (89/141; 63.1%) spermatozoa. Of the lambs resulting from embryos produced with sex-sorted spermatozoa, 86/93 (92.5%) were born of the predicted sex. This study demonstrated for the first time that pre-sexed offspring derived from superovulated sheep can be produced following transfer of embryos. Furthermore, sex-sorting by flow cytometry did not compromise the in vivo fertilizing capacity of ram spermatozoa in superovulated sheep, nor did it affect the quality or survivability of the resultant embryos.     

Pattern of loss of spermatozoa from the vagina of the ewe

In a series of experiments spermatozoa were inseminated blindly into the vagina of ewes and then recovered at varying times after insemination. Most of the spermatozoa inseminated were lost by drainage through the vulva. The rate of loss was not affected by the motility of spermatozoa or oestrous state of the ewe. Initially after insemination the loss was not rapid with 82% of the insemination 18% of spermatozoa remained and by 12 h 10% remained. Spermatozoa were removed from the vagina during withdrawal of the penis after intromission and the extent of this loss varied between rams and with the volume of semen already in the vagina. Up to half the inseminate was lost in this way when there was 0.5 ml of semen in the vagina but only 11% was lost when the volume of inseminate was 0.1 ml. The unavoidable loss of spermatozoa may influence the quantity available for fertilizing ova.     

Porcine sperm zona binding ability as an indicator of fertility

The escalated use of artificial insemination in swine has increased the importance of determining fertility of a semen sample before it is used. Multiple laboratory assays have been developed to assess fertilizing potential but they have yielded inconsistent results. This experiment sought to determine the relationship between in vitro competitive zona binding ability and in vivo fertility based on heterospermic inseminations and paternity testing. The zona pellucida binding ability and fertility of sperm from 15 boars was assessed by comparing sperm from one boar with sperm from other individual boars in a pairwise fashion using four ejaculates. The relationship of zona binding ability to the mean number of piglets sired per litter for each boar as well as historic fertility data (litter size and farrowing rate) was assessed. The in vitro competition assay consisted of labeling sperm from each boar of the pair with a different fluorophore and incubating an equal number of sperm from each boar in the same droplet with porcine oocytes. The competitive assay was highly effective in ranking boars by zona binding ability (R2=0.94). Paternity testing using microsatellite markers was used to determine the mean number of piglets sired per litter for each boar during heterospermic inseminations. The pairwise heterospermic insemination assay was effective in ranking boar fertility (R2=0.59). Using historical data from these boars, average litter size and farrowing rate were correlated (r=0.81, p<0.001). However, zona binding ability was not significantly correlated with historic farrowing rate data or historic average litter size. Boar sperm zona binding ability was also not correlated significantly with the mean number of piglets sired per litter following heterospermic insemination. But the number of piglets sired by each boar was related to a combination of zona binding ability, sperm motility, normal morphology, acrosomal integrity, and the presence of distal droplets (R2=0.70). These results suggest that zona binding ability is not an accurate predictor of fertilizing ability when used alone; however, when coupled with other sperm assessments, fertility may be predicted successfully.     

Sperm morphology is better in the second ejaculate than in the first in domestic cats electroejaculated twice during the same period of anesthesia

Several species produce ejaculates of inferior quality after a period of sexual abstinence, but the frequency of semen collection has thus far not been shown to affect sperm morphology in felids. The aim of this study was to determine whether sperm morphology and motility would differ between 2 ejaculates collected from the same cat within a short interval. Fifteen male domestic cats were anesthetized and then electroejaculated twice, with a 5- to 10-min interval between treatments. A standardized electroejaculation regimen was used with 80 stimuli, from 2 to 5 V, for each ejaculate. The first ejaculates contained significantly higher (P < 0.05) proportions of distal droplets, coiled tails and immotile spermatozoa than the second ejaculates, which contained significantly higher proportions of morphologically normal spermatozoa (40.9 vs 54.6%) but a lower sperm count (39.0 x 10(6) vs 5.2 x 10(6)). The higher proportions of defective spermatozoa and the lower motility in the first ejaculate than in the second were probably due to the aging of spermatozoa in the epididymis. These results show that the second ejaculate collected within a short interval has better sperm morphology and motility than the first and that this should be considered when evaluating semen quality in the domestic cat and when collecting cat semen to be used for artificial insemination or to be frozen for storage.     

Quality and freezing qualities of first and second ejaculates collected from endangered Gulf Coast Native rams

The Gulf Coast Native sheep, or Louisiana Native sheep, is an endangered previously feral domestic sheep population of European origin that has been under natural selection pressure for reproductive survival in their transplanted range while roaming in the southern Gulf Coast Region of the United States. This sheep population has an increased natural resistance to internal parasites, breeds year-around and has a greater percentage of live lambs as compared with other breeds of sheep raised in similar environments. To preserve the genetic diversity of this important feral sheep population, semen was collected by electro-ejaculation and subjected to cryopreservation for subsequent storage in a genome resource bank. Unrelated rams (n=5) were collected 3 days-a-week, allowing at least 2 days of rest between collections. Two ejaculates were obtained from each ram per collection day, with the second collection conducted 10min after the first ejaculation. Semen was processed using the standard Salamon cryopreservation procedure in a Tris-yolk-glycerol extender, frozen in 0.5ml plastic straws using liquid nitrogen (LN(2)) vapor and stored in LN(2). Each ejaculate was evaluated for volume, sperm concentration/ml (x10(9)/ml), number of spermatozoa/ejaculate (x10(9)), sperm progressive motility (%) for pre-cooled semen, cooled semen and semen after thawing. For the five rams, each semen variable for the first ejaculate was compared with that of the second ejaculate collected 10min later. The mean semen volume, sperm concentration and number of spermatozoa per ejaculate obtained from the first ejaculate were significantly greater (P< or =0.01) than those of the second ejaculate (comparisons being 1.62 and 1.06; 3.2 and 1.5; 5.4 and 1.8, respectively). Overall, the mean motility of pre-cooled (22 degrees Celsius), cooled (5 degrees Celsius) and frozen (-196 degrees Celsius) post-thawed spermatozoa was less (P< or =0.01) in the first ejaculate (71.5, 64.8 and 34.1%, respectively) compared with that of the second ejaculate (75, 72.4 and 44.1%, respectively). Conversely, no differences were detected in loss in the percent progressive motility of sperm from cooled sperm to post-thaw sperm from the first and second ejaculates. In summary, our findings suggest sperm collected during the second ejaculate 10min after the first ejaculate of rams survives thawing with a greater rate of progressive motility than that of the first ejaculate. The ability to collect two consecutive ejaculates in a short period by electro-ejaculation could be valuable for gamete resource banking and preserving genetic diversity of the Gulf Coast Native sheep.     

Breed differences in competitive indices of Holstein and Jersey bulls and their association with sperm DNA fragmentation index and plasma membrane integrity

The objective was to evaluate the relationship of a competitive index (CI) determined by heterospermic performance and post-thaw semen quality of the same stored ejaculates. Semen from multiple ejaculates collected in succession from each bull (four Holstein and four Jersey) was pooled. Heterospermic doses (20x10(6)/straw) were made to obtain all possible Holstein-Jersey combinations (16 two-bull combinations) and contained 20x10(6) sperm/mL/bull. Cows at two University dairy farms were inseminated on observed or synchronized estrus. The sire of calves (N=460) were determined and a CI was determined for each bull (based on the number of calves sired). Prior to preparation of the heterospermic doses, a sub-sample of semen from each bull was taken, processed, frozen, and stored concurrently with heterospermic samples. Post-thaw semen samples (homospermic) from each bull were assessed for: sperm morphology, acrosome integrity, sperm motility parameters assessed by computer assisted sperm analysis (CASA), flow cytometry analysis of DNA Fragmentation Index (DFI), and Plasma Membrane Integrity (PMI). Heterospermic performance of Holstein bulls was superior to that of Jersey bulls. The DFI was negatively correlated to CI (r=-0.87; P<0.001), whereas the PMI (r=0.87; P<0.001) and total progressive motility (r=0.74; P<0.05) assessed by CASA were positively correlated to CI. In multivariate regression models, the DFI and PMI accounted for 87% variance in competitive index. In conclusion, bulls with less DFI and higher PMI had higher probabilities of siring calves.     

Effects of age and environmental factors on semen production and semen quality of Austrian Simmental bulls

More than 90% of the breeding stock of Austrian dual purpose Simmental cows is artificially inseminated. Knowledge of factors affecting sperm production and semen quality is of importance with regard to reproductive efficiency and thus genetic improvement as well as for the productivity and profitability of AI centers. Hence, semen data from two Austrian AI centres collected in the years 2000 and 2001 were evaluated. In total, 3625 and 3654 ejaculates from 147 and 127 AI bulls, respectively, were analysed regarding ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. Effects accounted for were the bull (random), age of bull, collection interval, number of collection on collection day, bull handler, semen collector, temperature on day of semen collection, in the course of epididymal maturation (average temperature of days 1-11 before collection) and during spermatogenesis (average temperature of days 12-65 before collection). Age of bull significantly affected all traits (P<0.01 to P<0.001) except motility score in center 2. Ejaculate volume and total number of spermatozoa increased with age of bull while sperm concentration was lower in higher age classes (center 1). The collection team was also found to significantly influence semen quality traits. With increasing collection interval ejaculate volume and total number of spermatozoa increased significantly (P<0.05 to P<0.001) while collection intervals between 4-9 days and 1-6 days were superior with regard to sperm concentration and percentage of viable spermatozoa, respectively (P<0.10 to P<0.001). First ejaculates were superior with respect to ejaculate volumes, sperm concentrations and total number of spermatozoa per ejaculate (P<0.001). Temperature, either on day of semen collection or during epididymal maturation or spermatogenesis, had important but inconsistent effects on semen production and sperm quality. Overall, however, ambient temperatures in the range of 5-15 degrees C were found to be optimal for semen production.     

Relationships among lipid peroxidation, glutathione peroxidase, superoxide dismutase, sperm parameters, and competitive index in dairy bulls

Sperm membranes contain high concentrations of polyunsaturated fatty acids that are highly susceptible to oxidative damage that interferes with fertilization ability. The objective of this study was to determine associations among lipid peroxidation (thiobarbituric-acid-reactive substance concentration), antioxidant enzymatic activities in frozen spermatozoa, and competitive indices. Semen from multiple ejaculates collected in succession from each bull (four Holstein and four Jersey) was pooled. Heterospermic doses (20x10(6)sperm/0.5mL straw) were made to obtain 16 Holstein/Jersey combinations (equal number of sperm from each bull). Cows were inseminated on observed or synchronized estrus. The sire of calves (N=460) was determined; based on the number of calves sired, a competitive index was obtained for each bull. Prior to preparation of the heterospermic doses, a sub-sample of semen from each bull was taken, processed, frozen, and stored concurrent with heterospermic samples. After thawing, these homospermic samples were assessed for lipid peroxidation, superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, DNA fragmentation index (DFI), plasma membrane integrity (PMI), and total progressive motility (assessed by CASA). Sperm lipid peroxidation and the competitive index were negatively correlated (r=-0.78; P<0.05), the DFI and sperm lipid peroxidation were positively correlated (r=0.86; P<0.001), and there were negative correlations (P<0.05) for sperm lipid peroxidation and both PMI and total progressive motility (r=-0.78 and -0.83, respectively). There was neither significant association between SOD activity and competitive index, nor between GPx activity and competitive index. In conclusion, bulls with lower sperm lipid peroxidation had higher chances of siring calves; this was attributed to the deleterious effects of lipid peroxidation on sperm plasma membrane integrity and sperm DNA, which may reduce sperm fertilizing potential.     

La spermiologie: une discipline très particulière

Spermiology considers the ejaculate as the sole participation of man in fertilization. Spermiology as cognitive science, studies the components of the fertilizing ability of semen and spermatozoa and its disturbances. Spermiology as functional investigation, evaluates the fertility of individuals by analysing their ejaculates. Interventional spermiology handles sperm in the scope of medically Assisted Procreation. Spermiology is practiced in a constraining ideological and legal context.     

Some factors influencing the interaction of ram spermatozoa with zona-free hamster eggs

Variability in the interaction of ram spermatozoa with zona-free hamster eggs was recorded not only amongst individual males but also between the first and second ejaculates of the same male collected 30 min apart. Fusion ability also differed according to the conditions of gamete mixing. This ability decreased after in-vitro storage of undiluted ejaculates at room temperature but lasted for 48-192 h. The kinetics of sperm-egg fusion during the time of gamete incubation varied not only with the time of sperm storage in vitro but also with the ejaculate. When the semen was frozen, the ability of the spermatozoa to fuse was markedly reduced.     

Development of macroscopic and microscopic characteristics of ejaculates from Chios, Serres and Karaguniki breed lambs

The developmental characteristics in the volume of the ejaculate, motility (percentage of motile spermatozoa) and grade of motility (vitality), density, total number of spermatozoa per ejaculate and percentage of dead (stained) and of morphologically abnormal spermatozoa were studied in 14-to 46-week-old lambs of the Chios (n=10), Serres (=10) and Karaguniki (n=10) breeds, born in October 1984. The first appearance of spermatozoa in the ejaculate of the Chios, Serres and Karaguniki lambs occurred at 20.1+/-0.31 (x +/-SEM), 20.4+/-0.37 and 20.6+/-0.54 weeks of age, respectively, when the lambs had attained a body weight of 36.4+/-, 36.5+/-0.70 and 34.9+/-0.99 Kg, respectively. The volume of the ejaculate, the motility and the grade of motility of spermatozoa increased at a rapid rate up to the age of 32 weeks, when the relevant values were the same as those found in the adult animal. Density of the semen and the total number of spermatozoa per ejaculate increased at a slower rate up to the age of 46 weeks, while the percentages of dead (stained) and of morphologically abnormal spermatozoa decreased significantly between 20 and 32 weeks of age. It is concluded that the quality of the semen at the time when the first spermatozoa appear in the ejaculate is not satisfactory, but it improves in the course of the ensuing 2 to 3 months. The optimal age at which the lambs may be used for artificial insemination are 32, 36 and 34 weeks for the Chios, Serres and Karaguniki breeds, respectively.     

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.     

Time required for spermatozoa to remain in the vagina of the ewe to ensure conception

Oestrous ewes (N = 202) were inseminated with 0.1 ml of semen containing 500 X 10(6) motile spermatozoa and the spermatozoa were flushed from their vagina either immediately or 0.25, 0.5, 1, 2, 4 and 8 h after insemination. Pregnancy was determined by returns to service and laparoscopy. Some ewes became pregnant (10.71%) after spermatozoa had been flushed from the vagina only seconds after insemination and about 40% of ewes became pregnant after spermatozoa had been in the vagina for 15 min. Maximum conception (55%) was achieved when spermatozoa had been in the vagina for at least 2 h. It was concluded that the losses of spermatozoa that occur from the vagina will not influence the chance of a ewe conceiving because sufficient spermatozoa to ensure a normal conception move up the reproductive tract before large losses from the vagina take effect.