Fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with or without Equex STM paste

Twenty-five bitches were artificially inseminated with semen that was frozen-thawed using an egg yolk-Tris-glucose-citrate extender containing 5% glycerol with, or without the addition of 0.5% Equex STM Paste. Semen was collected on 2 occasions from 11 dogs, pooled, and evaluated for sperm motility, morphology and plasma membrane integrity. Each pool was then divided in 2 parts, diluted with 1 of the 2 extenders, and frozen in 0.5-mL straws. In the bitches, plasma progesterone was assayed daily during late proestrus and estrus. Artificial insemination (AI) was performed twice on Days 3 and 5 after the estimated LH peak. For each insemination, 200x10(6) spermatozoa were used. Ten bitches were inseminated with semen frozen without Equex: In 5 females, semen was deposited transcervically into the uterus with the aid of a fiberoptic endoscope and a urethral catheter, while the remaining 5 bitches were inseminated in the cranial vagina using a Norwegian catheter. Fifteen bitches were inseminated with semen frozen-thawed with Equex: Two groups of 5 bitches were inseminated according to the techniques described above, while 5 bitches were inseminated vaginally using the Osiris catheter. Pregnancy was diagnosed and the number of fetuses counted by ultrasound examination. Post-thaw, spermatozoa frozen with Equex tended to have higher total and progressive motility and to survive longer in vitro than when the extender without Equex was used. Spermatozoal concentration, age of the bitches, duration of heat and estrus, and progesterone concentration at LH peak and at the first and second AI did not differ among the 5 groups. The overall pregnancy rate of 84% (21/25) was close to what can be expected from well controlled natural matings. For both freezing extenders tested, 5/5 bitches were pregnant after uterine deposition of semen and 4/5 were pregnant when semen was deposited in the anterior vagina using the Norwegian catheter. With the Osiris catheter, 3/5 inseminations resulted in a pregnancy. No significant differences in pregnancy rate or number of fetuses were found between groups, site of deposition or freezing extender.     

Intravaginal insemination of bitches with fresh and frozen-thawed semen with addition of prostatic fluid: use of an infusion pipette and the Osiris catheter

One hundred fifty-two bitches of seven breeds were vaginally inseminated with fresh or frozen-thawed semen of 10 stud dogs of respective breeds. The semen was supplemented with prostatic fluid before insemination. In experiment 1 bitches of each breed were randomly assigned to three treatment groups, consisting of 29 females (group 1), 33 females (group 2) and 32 females (group 3). In group 1 bitches were inseminated into vagina with fresh semen using a bovine infusion pipette. In group 2 bitches were inseminated into vagina with fresh semen using the Osiris catheter. In group 3 bitches were inseminated with frozen-thawed semen with the Osiris catheter. The number of sperms in each insemination dose was adjusted to 300 x 10(6). In experiment two bitches were randomly assigned to two treatment groups, consisting of 30 females (group A) and 28 females (group B). In group A bitches were inseminated with fresh semen, whereas in group B with frozen-thawed semen. Osiris catheter was used in both groups. The total number of sperms was adjusted to provide 250 x 10(6) of progressively motile spermatozoa in each insemination dose. In experiment 1 the pregnancy rates/whelping rates were 86.2/82.8%, 81.8/81.8% and 59.4/59.4% for groups 1, 2 and 3, respectively. The differences between group 1 and 3 were statistically significant (p < 0.05). The litter sizes at birth/litter sizes at weaning were 5.8+/-2.3/5.4+/-2.0, 6.3+/-1.4/5.7+/-1.0 and 3.9+/-1.2/3.5+/-1.5 in groups 1, 2 and 3, respectively. The litter size at birth and at weaning was reduced (p < 0.05) when frozen-thawed semen was used for insemination (group 3). There were not significant (p > 0.05) differences in the litter size between groups 1 and 2. In experiment 2 pregnancy rates/whelping rates and litter sizes at birth/litter sizes at weaning were 86.7/86.7%, 60.7/57.1% (p < 0.05) and 6.1+/-1.6/5.7+/-1.7, 4.0+/-1.4/3.8+/-1.4 (p < 0.05) in groups A and B, respectively. This study shows that results of AI with a fresh semen using a bovine infusion pipette and the Osiris catheter are equivalent. The results of the use of the Osiris catheter for vaginal insemination of frozen-thawed dog semen extended with prostatic fluid after thawing are not encouraging. The pregnancy rate, whelping rate and litter size are reduced when frozen-thawed, prostatic fluid-supplemented semen is vaginally deposited using the Osiris catheter.     

Influence of extender, cryoperservative and seminal processing procedures on postthaw motility of canine spermatozoa frozen in straws

Experiments were conducted to evaluate two extenders (egg-yolk Tris and egg-yolk lactose), varying concentrations of two cryopreservatives (glycerol and dimethyl sulfoxide), and rates for cooling to 5 degrees C, cooling from 5 to -100 degrees C, and warming for canine spermatozoa packaged in 0.5-ml French straws. At optimal concentrations of glycerol, egg-yolk Tris extender was superior to egg-yolk lactose in preserving spermatozoal motility. Addition of dimethyl sulfoxide, alone or in combination with glycerol in either extender, was not beneficial to spermatozoal survival after thawing. Canine spermatozoa withstood a range of cooling and equilibration times with no detrimental effect on spermatozoal motility prior to freezing. However, there were differences in spermatozoal motility immediately after thawing; these differences were variable, resulting in a cooling time by equilibration time interaction. Spermatozoal motility after thawing was best preserved by freezing in egg-yolk Tris extender containing 2-4% glycerol, using a moderate rate of cooling from 5 to -100 degrees C (-5 degrees C/min from 5 to -15 degrees C, then -20 degrees C/min from -15 to -100 degrees C). Three of 12 bitches inseminated intravaginally with semen frozen using this protocol became pregnant.     

Long-term motility and fertility conservation of chilled canine semen using egg yolk added Tris-glucose extender: in vitro and in vivo studies

The effects of medium exchange on motility parameters of chilled canine semen preserved in egg yolk Tris-glucose (EYTG) extender were analyzed over a 27-d period. Semen extender was exchanged at three time points (Days 11, 21 and 27) after collection, when motility parameters were demonstrated to significantly decrease from parameters observed at semen preparation (Day 0) or at day of previous extender exchange. In the absence of medium exchanges, motile spermatozoa were observed up to Day 16 (mean +/- S.D. 1.5 +/- 0.3% of motile spermatozoa). A stimulation of the different semen motility parameters was observed after extender exchange. Semen extender exchange at Day 11 allowed conservation of motility until Day 21, compared to 16 d in the absence of extender exchange. At Day 21, when spermatozoa appeared immobile or dead, a second extender exchange was performed, allowing the extension of motility conservation up to Day 27. The third extender exchange, performed at Day 27, was no longer associated with motility stimulation. Glucose content in the medium decreased slowly over time; a concomitant decrease in pH was also observed. No changes in osmolarity were observed over time. To verify the fertility of long-term conserved chilled semen, two groups of 10 bitches were inseminated either once (Group 1) or twice at 48-h intervals (Group 2) intra-vaginally with semen conserved chilled for a mean of 9 +/- 1.8 d. Out of the 10 bitches inseminated once, 5 became pregnant, versus 7 in the group of animals inseminated twice. The present study reports the possibility to extend the conservation of chilled canine semen up to 3 wk with conservation of good fertility for at least 10 d. The role of energetic substrate and pH alteration is postulated and the classically accepted relation of semen motility/viability is raised.     

Equine spermatozoal motility and fertility associated with the incorporation of d-(+)-mannose into semen extender

Mannose is capable of decreasing bacterial attachment to the uterine mucosa in mares. Bacteria gain entry into the mare's uterus during breeding; therefore, a practical method to deliver mannose to the uterus is to incorporate it into semen extenders. The effect of mannose on spermatozoal motility and subsequent sperm fertilizing capability is unknown. The present study evaluated progressive spermatozoal motility in semen extender formulations incorporating mannose and assessed the fertility of mares inseminated with a mannose-containing semen extender. In Experiment 1, progressive spermatozoal motility in extender mixtures containing 0 mannose (control), 25, 37 or 49 mg/mL mannose was evaluated at 20 degrees C or 5 degrees C holding temperatures for 0, 12, 24 and 48 h post-dilution. Measures were repeated three times using five stallions of proven fertility. High concentrations of mannose in the extender affected progressive motility beyond the time and temperature effects noted in the controls. Extender containing only mannose sugar (49 mg/mL) displayed an immediate depression in progressive motility compared with controls (45.5% versus 62.9%, respectively; P<0.001). The 37 mg/mL mannose extender had a less dramatic decrease in motility (P<0.05) and only after storage at 5 degrees C for > or =12h (48.7% versus 58.0%, respectively). Extender with 25 mg/mL mannose performed no differently than the control formulation under all conditions. In Experiment 2, two groups of mares (n=11 each) were inseminated with 500 x 10(6) progressively motile spermatozoa extended in a traditional skim milk (control) extender or the 37 mg/mL mannose extender preparation. A single-cycle pregnancy rate of 72% was achieved by both groups. Present data suggest that a semen extender containing up to 37 mg/mL mannose could maintain motile spermatozoa for on-farm use and 25 mg/mL mannose concentrations preserved motility during long-term cooling. Likewise, sperm extended with up to 37 mg/mL of mannose had the same fertilizing capability as sperm in traditional extender mixtures.     

Comparisons between three different extenders for canine intrauterine insemination with frozen-thawed spermatozoa

Ejaculates from 7 dogs were obtained on the same day and were pooled. This pooled semen was separated into 3 equal fractions and processed simultaneously, the only difference being in the extender used for freezing. The extenders were laiciphos (containing laiciphos, egg yolk, distilled water and glycerol- Group 1); Tes/Tris (containing Tes/Tris, egg yolk, distilled water and glycerol- Group 2); and biociphos (containing biociphos with glycerol in it, egg yolk and distilled water- Group 3). Spermatozoa were conditioned in 0.5ml French straws and presented normal characteristics before freezing and after thawing. The sperm concentration of the pooled was 683 x 10(6) sperm/ml; sperm motility was above 95%, the percentage of live spermatozoa was above 95% and was of good quality and mobility. Characteristics of the spermatozoa after thawing were the same for spermatozoa frozen with laiciphos and Tes/Tris. Mean sperm concentration was 201.5 +/- 4.95 x 10(6) sperm/ml, sperm motility was 65%, the percentage of live spermatozoa was 80% and the quality of motility.was good. Spermtozoa frozen with biociphos had the following post-thaw characteristics: sperm concentration was 201 x 10(6) sperm/ml, sperm motility was 50%, the percentage of live spermatozoa was 78% and the quality of mobility was medium. Abnormalities were less than 15% for all spermatozoa after thawing. Intrauterine artificial inseminations were performed by laparoscopic intrauterine insemination twice at Days 3 and 5 after the estimated LH peak in 15 normally cyclic Beagle bitches (5 per group) presenting normal hormonal profiles. There were no differences between groups. The females were inseminated with 1.0 ml of spermoatozoa (concentration of 200 x 10(6) sperm/ml) diluted with 1.0 ml of extender. A 60% pregnancy rate was obtained in bitches inseminated with frozen-thawed spermatozoa extended with laiciphos or Tes/Tris and 100% in bitches inseminated with spermatozoa extended with biociphos. Females inseminated with laiciphos, Tes/Tris and biociphos had a mean litter size of 5 +/- 2.6, 3 +/- 1 and 3.4 +/- 1.3 pups, respectively. This study demonstrated that post-thaw assessment of sperm characteristics is not the best technique for evaluating sperm fertility after freezing or for assessing different semen extenders.     

Pregnancy and conception rate after two intravaginal inseminations with dog semen frozen either with 5% glycerol or 5% ethylene glycol

The primary goal of this study was to compare the effects of 5% ethylene glycol (EG) and 5% glycerol (G) on fertility of frozen–thawed dog semen following intravaginal insemination. The sperm-rich fraction of the ejaculate of three male dogs was collected, pooled and divided into two aliquots, and then frozen with a Tris–glucose–egg yolk–citric acid extender containing either 5% G or 5% EG. A total of 10 bitches were inseminated twice, five with G-frozen–thawed semen and five with EG-frozen–thawed semen; intravaginal inseminations were performed the 4th and the 5th day after the estimated LH peak; four straws, thawed in a 37 °C water bath for 1 min and diluted in a Tris buffer, were used for insemination (200 × 10⁶ spermatozoa); the insemination dose was introduced in the cranial vagina of the bitch using a sterile plastic catheter. Ovariohysterectomy was performed in all bitches between days 29 and 31 after the calculated LH surge, and pregnancy status, and the number of conceptuses and corpora lutea were recorded. All bitches were pregnant. Neither the number of conceptuses, nor the ratio of conceptuses to corpora lutea (conception rate) was significantly different between groups. In this first screening, with a limited number of bitches, EG-frozen semen did not show a higher fertility than G-frozen semen when used for two intravaginal inseminations. Irrespective of the semen used, conception rate was 0.50.     

Assessment of fresh and stored duck spermatozoa quality via in vitro sperm-egg interaction assay

An in vitro sperm-egg interaction assay was used to measue the quality of duck spermatozoa in fresh and stored semen. The inner perivitelline layer (IPVL), which had been separated from laid duck eggs, was incubated with spermatozoa in vitro. The number of points of sperm hydrolysis in the IPVL in vitro was logarithmically correlated with the fertility of the eggs laid by inseminated females, for both fresh semen (r = 0.85, P < 0.001) and stored semen at 5 degrees C for 24 h (r = 0.84, P < 0.001). After semen storage, the ability of spermatozoa to hydrolyze the IPVL decreased by 67.4% compared with the values for fresh semen, whereas egg fertility and sperm motility decreased by 47.8% and 15.2%, respectively. These results suggest that the in vitro sperm-egg interaction assay accurately reflects the fertilizing ability of fresh and stored duck spermatozoa and detects spermatozoal damage due to semen storage more sensitively than motility or fertility tests.     

The effect of x-radiation upon the quality and fertility of stallion semen

The exposure that stallion semen might receive during examination using an airport x-ray security screening system was found to be between 0.5 and 1.0 micro Sieverts (μSv). Ejaculates from 2 stallions were diluted 1:4 (volume:volume) using a nonfat dried milk-glucose extender. A total of 6 ejaculates from each stallion was collected, and each ejaculate was divided into 3 aliquots and these were then exposed to x-radiation at a dose of 0, 1.0, or 10.0 (μSv. Semen quamy was examined immediately post exposure, and the aliquots were then placed into a water bath at 37 °C, after which sample longevity was evaluated.In a second trial, 3 groups of 8 pony mares were inseminated with semen that had been exposed to x-radiation at doses of 0, 1.0, or 10.0 μSv. An entire ejaculate was irradiated and inseminated into each mare on one occasion during estrus, based upon ultrasonographic evaluation of the reproductive tract.After exposure to x-radiation there were no differences among the 3 treatment groups for spermatozoal motility, morphology, or longevity. The 14-d pregnancy rates for the 3 treatment groups were 0 μSv (7 mares), 1.0 μSv (8 mares), and 10.0 μSv (7 mares). One mare (0 (μSv) aborted at 65 d of pregnancy; 21 mares had a pregnancy of normal length, with each delivering a foal at term, although 1 foal died at parturition (1.0 μSv).These findings indicate that the exposure of stallion spermatozoa to x-radiation up to doses of 10 μSv does not have deleterious effects upon spermatozoal motility, morphology, longevity or fertility. The exposure received during examination using an x-ray security screening system is likely to be lower than this dose.     

Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage

The objective of this study was to determine if centrifugation and partial removal of seminal plasma would improve spermatozoal motility in semen from stallions whose whole ejaculates have poor tolerance to cooling and storage. Stallions were divided into two groups (n = 5/group) based on the ability of their extended semen to maintain spermatozoal motility after cooling and storage. Group 1 stallions ("good coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by < or = 30% of progressive motility prior to storage. Group 2 stallions ("poor coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by > or = 40% of progressive motility prior to storage. The sperm-rich portion of each ejaculate was divided into 4 aliquots. Two aliquots underwent standard processing for cooled transported semen and were examined after 24 and 48 h of cooling and storage in an Equitainer. The remaining two aliquots were diluted 1:1 with semen extender, then centrifuged at 400 x g for 12 min at room temperature. After centrifugation, approximately 90% of the seminal plasma was removed, and the sperm pellet was resuspended in extender to a final concentration of 25 to 50 x 10(6) sperm/mL. These aliquots were then packaged as for the non-centrifuged aliquots and examined after 24 and 48 h of storage. The spermatozoal motion characteristics in fresh semen and after 24 and 48 h of cooling and storage was determined via computer-assisted semen analysis. Centrifugation and partial removal of seminal plasma increased the percentage of progressively motile spermatozoa and limited the reduction in progressive spermatozoal motility of "poor cooling" stallions after 48 h of cooling and storage. Results of this study indicate that centrifugation and partial removal of seminal plasma is beneficial for stallions whose ejaculates have poor tolerance to cooling and storage with routine semen dilution and packaging techniques, especially if the semen is stored for > 24 h.     

Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa

A multifactorial study analyzed the effects of freezing method, cryoprotective diluent, semen to diluent ratio, and thawing velocity on post-thaw motility, progressive status, and acrosomal integrity of ram spermatozoa. Although semen to diluent ratio (1:3 vs 1:6, v/v) had no effect (P greater than 0.05), overall post-thaw spermatozoal viability was highly dependent on freezing method and cryoprotectant. Improved results were obtained by freezing semen in 0.5-ml French straws compared to dry ice pelleting. Manually freezing straws 5 cm above liquid nitrogen (LN2) was comparable to cooling straws in an automated, programmable LN2 unit. Of the two cryoprotective diluents tested, BF5F (containing the surfactant component sodium and triethanolamine lauryl sulfate) yielded approximately 50% fewer (P less than 0.05) spermatozoa with loose acrosomal caps compared to TEST. Thawing straws in a water bath at a higher velocity (60 degrees C for 8 sec) had no effect (P greater than 0.05) on spermatozoal motility, progressive status ratings, or acrosomal integrity when compared to a lower rate (37 degrees C for 20 sec). For the TEST group, thawing pellets in a dry, glass culture tube promoted (P less than 0.05) percentage sperm motility at 3 and 6 hr post-thawing, but for BF5F diluted semen this approach decreased the % of spermatozoa with normal apical ridges. The results suggest that the poor fertility rates often experienced using thawed ram semen likely result not only from reduced sperm motility, but also from compromised ultrastructural integrity. This damage is expressed by an increased loosening of the acrosomal cap, a factor which appears insensitive to freezing method but markedly influenced by the cryoprotective properties of the diluents tested.     

Fertility in bitches artificially inseminated with extended, chilled semen

Sixteen bitches were artificially inseminated with either fresh, 24 h-chilled or 48 h-chilled extended semen over 38 estrous cycles. A commercial system for extending, chilling and transporting semen commonly used in the equine industry was used Pregnancy rates and litter sizes of the bitches inseminated with extended, chilled semen (19/20, 95%; litter size = 7.1) were not significantly different from those observed in bitches inseminated with fresh semen (17/18, 94%; litter size = 7.2; P > or = 0.89). These results show that a commercial system for extending, chilling and transporting equine semen is an attractive and efficient method of shipping canine extended chilled semen.     

Development of a chemically defined ram semen diluent (RSD-1)

A chemically defined ram semen diluent (RSD-1) has been developed. RSD-1 maintained spermatozoal motility of diluted semen containing approximately 800 million spermatozoa ml⁻¹ during cooling to 15°C and its storage for 1 h. Motility was further maintained when the cooled semen was diluted to 100 million spermatozoa ml⁻¹ and incubated at 38°C for about 24 h. In contrast, a conventional milk-based diluent supported motility for less than 6 h at 38°C. Spermatozoal motility was influenced by the buffering capacity, osmolarity and the presence or absence of macromolecules and calcium in the chemically defined diluent. Among the organic buffers tested, MOPS (3-(N-morpholino)propanesulphonic acid) had a marked influence on the maintenance of spermatozoal motility. The presence of MOPS also overcame the detrimental effects of 2 mM calcium in Krebs Ringer improved (KR-I) buffer.     

Motility and fertility of stallion spermatozoa isolated in bovine serum albumin

Semen from three mature stallions was used in an attempt to isolate a population of highly motile spermatozoa. An ejaculate of semen, collected from each stallion at 7-day intervals for 35 days, was evaluated for percentage of motile spermatozoa and rate of progressive motility (scale 1 to 4). Two milliliters of semen were layered over 6 ml of 3% bovine serum albumin (BSA) in 13 × 125 mm columns at room temperature (RMT) or in a warm water bath (WB). After a 30-minute separation period, the top semen layer and the upper and lower halves of the BSA fraction were separately withdrawn from columns and reevaluated. In both the RMT and WB separation columns, percent motile spermatozoa and progressive motility decreased in the top semen fraction as compared to initial values for these parameters. Percentage of motile spermatozoa in the lower BSA fractions increased (P<.01) to 58.7 and 65.7 following separation at RMT and in a WB, respectively. Also, there was a significant increase in rate of progressive motility rate for spermatozoa in the lower BSA fraction of both the RMT and WB treatments.In a second experiment 30 Quater Horse mares were artificially inseminated to compare fertility of spermatozoa isolated in BSA with raw semen diluted with either Tyrode's solution or BSA. The pregnancy rate for 10 mares inseminated with 100 × 10⁶ live isolated spermatozoa was not different from that of control mares inseminated with the same number of live untreated spermatozoa. Foaling rates were 70, 40 and 60% for the isolated, Tyrode and BSA treatment groups respectively.     

Effect of different diluents on goat semen fertility

Skim milk (SM) is considered to be the most widely employed extender for goat sperm used for artificial insemination (AI). However, the fertilizing life span of sperm stored in milk or milk-based extenders does not exceed 12h. Besides some seminal plasma components, such as a protein fraction from the goat bulbourethral gland secretion (SBUIII), interacts with some milk fractions and inhibits the spermatozoa motility. The aim of this study was to prolong the survival of buck semen and its fertility. Buck ejaculates were diluted to a final concentration of 100x10(6)spermatozoa/ml with three different diluents: SM, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPOL+hyaluronic acid (TEMPOL+HA). At 7h from dilution 42 goats were inseminated with semen diluted with SM (short-term semen) while after storage for 24h, 44 and 45 goats were inseminated with semen diluted with TEMPOL and TEMPOL+HA (long-term storage), respectively. At day 50 from AI the percentages of pregnant goats were 71.4% (30/42) with SM, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, with significant differences between SM and TEMPOL+HA. The kidding rate was 66.7% (28/42) with SM diluent, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, without significant differences among treatment groups. In conclusion, it is possible to maintain good fertility in goats after AI with semen stored for 24h in TEMPOL.     

Effect of storage temperature, cooling rates and two different semen extenders on canine spermatozoal motility

Ejaculates were collected form three mixed-breed male dogs daily for 3 d. The semen was diluted in either a nonfat dried milk solid-glucose (NFDMS-G) or egg yolk citrate (EYC) extender at a concentration of 25 x 10(6) sperm/ml. The diluted samples were exposed to three different storage temperatures (35, 22 and 4 degrees C). Three cooling rates (-1.0, -0.3 and -0.1 degrees C/min) were also investigated at the lowest storage temperature (4 degrees C). The semen was evaluated for total motility, progressive motility and velocity at 0, 6, 12, 24, 48, 72, 96 and 120 h after collection by two independent observers. Interactions between extenders, temperatures and time after collection were found for each of the variables. Nonfat dried milk solid-glucose diluent was superior to EYC (P<0.05) in preservating sperm motility parameters that were evaluated for most of the observations. The evaluated sperm motility parameters were also significantly superior (P<0.05) in semen stored at 4 degrees C than at 35 or 22 degrees C for most of the observations. The progressive motility and velocity of sperm in semen cooled at 4 degrees C in NFDMS-G were higher (P<0.05) at the fast and medium cooling rates (-1.0 and -0.3 degrees C) than at the slow cooling rate (-0.1 degrees C/min) at 24 and 72 h, and at 48 h, respectively. In conclusion, the present study suggests that canine spermatozoal motility is well preserved when a NFDMS-glucose extender is added to the semen and the semen is cooled at a medium or fast rate to a storage temperature of 4 degrees C. Additional studies are needed to evaluate the fertility of semen stored in this manner.     

Inhibition of motility of bovine, canine and equine spermatozoa by artificial vagina lubricants

The effects of four vaginal lubricants on progressive spermatozoal motility were evaluated. Neat semen was exposed to 0, 5, or 10% (w/v) of H-R, sterile K-Y, nonsterile K-Y or Maxilube lubricating jellies for 10 min at 37 degrees C and then extended to 10x10(6) spermatozoa/ml. Spermatozoal motility was evaluated after 0, 1, 2, 4 and 6 or 8 h of incubation at 37 degrees C. For bovine spermatozoa, sterile K-Y jelly at 10% suppressed motility (P<0.05), but nonsterile K-Y, H-R and Maxilube jellies had no effect. Maxilube was toxic (P<0.01) to canine spermatozoa and is not recommended for use during collection or insemination of canine semen. Exposure of equine semen to 10% H-R jelly had no effect on spermatozoal motility, whereas 10% sterile K-Y, nonsterile K-Y or Maxilube jellies suppressed (P<0.05) motility. For all three species, the new, nonsterile K-Y jelly was no more deleterious to spermatozoal motility than the old, sterile K-Y jelly, and H-R jelly also was satisfactory. Fertility tests are required to determine the effect of these products on fertility.     

Cooling rates,storage temperatures and fertility of extended equine spermatozoa

Two experiments were designed to evaluate cooling rates and storage temperatures for stallion spermatozoa extended in caprogen (CAP), Cornell University extender (CUE), heated skimmilk (SM) and a nonfat-dried milk solids glucose extender (NFDMS-glucose). In Experiment 1, each extender was evaluated in a separate but similar 4 × 4 × 6 factorial trial using two ejaculates from each of six stallions. Aliquots of 66 × 10⁶ spermatozoa were transferred to each of 16 coded tubes and extended to 6 ml with SM, CAP, CUE or NFDMS-glucose. Four tubes of extended semen were either plunged into 5C water or cooled at a rate of ?1.0, ?0.5, or ?0.2C/min. Within each treatment, one tube of extended semen was maintained at 20C, 15C, 10C or 5C. Progressive spermatozoal motility was estimated immediately after dilution (0 h) and at 4, 8, 12, 24 and 36 h. Regardless of extender, all three slower cooling rates were superior (P<0.05) to plunging to 5C; storage temperatures of 20C and 15C were superior (P<0.05) for maintaining spermatozoal motility. Experiment 2 was designed so that all extenders could be evaluated simultaneously. Since CUE resulted in an immediate depression of spermatozoal motility, it was not evaluated further. Semen was collected from 12 stallions and each ejaculate was extended in SM, CAP and NFDMS-glucose. Semen was cooled at ?1.0C/min and maintained at either 20C or 15C. Spermatozoal motility was assessed as in Experiment 1. Overall, the CAP and NFDMS-glucose extenders were superior (P<0.05) to SM for maintenance of spermatozoal motility. Storage at 20C or 15C resulted in similar (P>0.05) spermatozoal motility. Two fertility trials compared the use of SM and NFDMS-glucose extenders. Embryo recovery 6 days post-ovulation (Experiment 3) and pregnancy rate 50 days postestrus (Experiment 4) was similar (P>0.05) for mares inseminated with spermatozoa extended in SM or NFDMS-glucose.     

Effects of semen filtration and dilution rate on morphology and fertility of frozen gander spermatozoa.

Feces, urates or dirt originating from feathers often contaminate gander semen during collection, threatening its fertilizing ability. Seminal plasma used as a diluent has a similar effect, particularly on spermatozoa subjected to cryopreservation or short-term storage under refrigeration. The aim of the experiments was to evaluate the effects on spermatozoa motility, morphology and fertilizing ability after minimizing the influence of the contaminants by semen filtration or dilution prior to freezing. Pooled semen, collected twice a week from 9 White Italian ganders by dorso-abdominal massage, was divided into two parts. One sample was filtered and both were diluted in 1:1 or 1:0.5 (v/v) with EK diluent, equilibrated for 15 min at +4 degrees C, mixed with dimethyl-acetamide (DMA) in the final concentration 6% (v/v) and frozen to -140 degrees C in a computerized freezer, at a rate of 60 degrees C/min. In fresh and processed (filtered, freeze-thawed) semen were examined the spermatozoa motility and morphology, and fertilizing ability for freeze-thawed semen, both for unfiltered and filtered. In freeze-thawed semen no tangible differences due to experimental factors were observed in motility and percent of live spermatozoa in total. On average 35 to 42% of the spermatozoa survived the freezing process, but only 10 to 15% were normal, without any damage visible under the light microscope. The fertility of unfiltered freeze-thawed semen inseminated twice a week in a 0.2 mL dose (about 3 to 5 x 10(6) of live normal spermatozoa each) averaged 66.1% and hatchability of the set eggs 57.1 and 86.5% of the fertile eggs. The fertility obtained after the insemination with semen filtered prior to freezing was lower (64.3%), but hatchability was slightly higher (58.6 and 91.1% of set and fertile eggs, respectively). The duration of fertility for filtered semen was longer than that for unfiltered, 10 days after the last insemination the eggs were still fertile. The fertility results of freeze-thawed gander semen were very promising taking into consideration the small amount of inseminated live normal spermatozoa and it is possible to improve this result by increasing the number of spermatozoa in the insemination dose.     

Artificial insemination of sheep: Effects on fertility of number of spermatozoa inseminated and of storage of diluted semen for up to 18 hours at 5°C

Ram semen was prepared in a buffered glucose-saline solution containing 3% (v/v) egg yolk so that insemination doses of 25 or 100 million spermatozoa in volumes of 50 or 250 μl could be given per ewe at artificial insemination (AI). Fertility was significantly reduced by dilution and, within the treatments of diluted semen, significantly higher lambing rates followed the use of doses of 100 million spermatozoa. The volume of the AI dose had no significant effect on fertility.Of 945 inseminations performed using diluted semen, 388 were with samples that had been cooled to 5°C and stored chilled for 5 or 18 hr. The mean lambing result of 40% for freshly diluted semen was significantly higher than 31.6% and 30.2% for samples stored chilled for 5 and 18 hr respectively. Ewes inseminated with doses of chilled semen containing 25 million spermatozoa had a low lambing rate of 21.3%. The presence of 7.5% glycerol (v/v) in the diluent did not significantly affect the fertility of chilled semen.