Relationship between in vitro fertilisation of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen-thawed ram semen

No laboratory test exists that can reliably predict differences among rams in field fertility after artificial insemination (AI) with frozen-thawed semen. In vitro fertilisation (IVF) has been proposed as a method of predicting these differences. The objectives of this study were to evaluate whether IVF system could discriminate among rams of different fertility in vivo after AI using frozen-thawed semen. Also, to examine effects of lowering sperm concentration on discrimination power between rams used for IVF. The aim of Experiment 1 was to evaluate the effect of altering the sperm concentration from 2 x 10(6) to 0.03125 x 10(6) spermatozoa/mL on subsequent cleavage rate and blastocyst rate in vitro. In Experiment 2, six rams (three High and three Low in vivo fertility; average pregnancy rates of 37.6% and 21.8%, respectively) were compared for their fertilising ability in IVF. Spermatozoa from each of the six rams were added to ewe oocytes using a concentration of either 2 x 10(6) or 0.0625 x 10(6)/mL. There were six replicates with 25 oocytes per well and two wells per ram per replicate. Cleavage rate was monitored at 48 h post-insemination (p.i.) and blastocyst rate determined on Days 6-8 p.i. In Experiment 1, cleavage rate increased with increasing sperm concentration and blastocyst rate was not affected by sperm concentration on any day. When the six rams were tested using 2 x 10(6) spermatozoa/mL, no significant differences were found between High and Low fertility groups for cleavage rate or blastocyst rate on Days 6, 7, or 8 p.i. (P>0.05). When the experiment was repeated using 0.0625 x 10(6) spermatozoa/mL, no differences were found between High and Low group rams for blastocyst rate on any of Days 6, 7 or 8 p.i. (P>0.05). However, there was a significant difference between High and Low fertility rams for percentage of oocytes cleaved (16.4, S.E. 2.02%; P<0.01) and the correlation between fertility in vivo and cleavage rate in vitro was significant (P=0.013). Replicate of IVF was a source of significant variation for both cleavage rate and blastocyst rate and conditions need to be further controlled. However, we suggest that using a low concentration of spermatozoa (0.0625 x 10(6)/mL) for IVF may be a useful method for predicting field fertility of frozen-thawed ram semen.     

Effect of spermatozoal concentration and number on fertility of frozen equine semen

Information on the number of motile spermatozoa needed to maximize pregnancy rates for frozen-thawed stallion semen is limited. Furthermore, concentration of spermatozoa per 0.5-mL straw has been shown to affect post-thaw motility (7). The objectives of this study were 1) to compare the effect of increasing the concentration of spermatozoa in 0.5-mL straws from 400 to 1,600 x 10(6) spermatozoa/mL on pregnancy rate of mares, and 2) to determine whether increasing the insemination dose from approximately 320 to 800 million progressively motile spermatozoa after thawing would increase pregnancy rates. Several ejaculates from each of 5 stallions were frozen in a skim milk-egg yolk based freezing medium at 2 spermatozoal concentrations in 0.5-mL polyvinyl-chloride straws. Half of each ejaculate was frozen at 400 x 10(6) cells/mL and half at 1,600 x 10(6) cells/mL. Insemination doses were based on post-thaw spermatozoal motility and contained approximately 320 x 10(6) (320 to 400) motile spermatozoa or approximately 800 x 10(6) (800 to 900) motile spermatozoa. Sixty-three mares were assigned to 1 of 4 spermatozoal treatments (1--low spermatozoal number, low concentration; 2--low spermatozoal number, high concentration; 3--high spermatozoal number, low concentration; 4--high spermatozoal number, high concentration) and were inseminated daily. Post-thaw spermatozoal motility was similar for cells frozen at both spermatozoal concentrations (P > 0.1). One-cycle pregnancy rates were 15, 40, 28 and 33%, respectively, for Treatments 1, 2, 3 and 4. Packaging spermatozoa at the high concentration tended to increase pregnancy rates vs packaging at the low concentration (37 vs 22%; P = 0.095). Furthermore, when the lower spermatozoal number was used, there tended (P < 0.1) to be a higher pregnancy rate if spermatozoa were packaged at the higher concentration. There was no increase in pregnancy rates when higher numbers of motile spermatozoa were inseminated (27 vs 31%; P > 0.1). Based on these results, a single 0.5-mL straw dose containing 800 x 10(6) spermatozoa should be used and each insemination dose should contain approximately 320 x 10(6) motile spermatozoa. Fertility trials utilizing other freezing extenders are necessary before recommending a single 0.5-mL insemination dose for all freezing extenders.     

Viability and fertility of rabbit spermatozoa diluted in Tris-buffer extenders and stored at 15 degrees C

Artificial insemination (AI) in rabbits is not extensive in the breeding programs of the rabbit meat industry. A limiting factor is related to the semen preservation. In order to improve the use of AI, two experiments have been conducted to evaluate sperm viability and fertility of rabbit semen chilled and stored at 15 degrees C after dilution in Tris-based extenders. In Experiment 1, pooled semen samples were diluted 1:10 (semen/extender) in four different Tris-based extenders (Tris-citric-glucose (TCG), TES-Tris-glucose (TTG), Tris-citric-fructose (TCF) and TES-Tris-fructose (TTF)) and stored at 15 degrees C. Sperm viability was evaluated at 0, 24, 48, 72 and 96 h following dilution for total sperm motility (TSM), forward progressive motility (FPM), plasma membrane integrity (PMI) and acrosome integrity (NAR). Viability of spermatozoa declined with time of storage (P<0.05), irrespective of the extender used. There were interactions between extender and time of storage (P<0.05) in all viability parameters evaluated. After 96 h of storage, TCG provided the highest sperm viability (P<0.05) and TTG the lowest (P>0.05). In Experiment 2, a field trial was conducted at a commercial farm to evaluate the conception and farrowing rates of rabbit spermatozoa extended in TCG. After synchronization of oestrous and induction of ovulation, 3713 does with different physiological conditions (nulliparous, primiparous, lactating and re-breeding) were inseminated one time (15x10(6) sperm per doses) with semen stored at 0 (n: 1275), 24 (n: 1503) and 48 h (n: 935) at 15 degrees C. Overall conception and farrowing rates were 77.1+/-0.7 and 70.4+/-0.7, respectively, and the mean litter size was 7.6+/-0.1. Fertility results were unaffected by the time of semen storage (P>0.05). Regardless of time of semen storage, fertility results were affected by the physiological conditions of does (P<0.05). Nulliparous and lactating does showed the highest fertility and primiparous the lowest. In summary, these results indicate that Tris-buffer extenders are effective for preserving viability and fertilizing capability of rabbit spermatozoa stored at 15 degrees C.     

Influence of centrifugation and different extenders on post-thaw sperm quality of ram semen

Using a 2-step extension methodology to freeze ram semen, 2 freezing protocols (P1 and P2) and 3 extenders were evaluated in a split-sample experiment. The freezing protocols were tested in combination with Extenders A and B (Experiment 1), and B and C (Experiment 2). Protocol 1 included centrifugation before filling the straws to reconcentrate the diluted semen to a calculated sperm concentration of 800 x 10(6) cells/mL. Protocol 2 involved appropriate ejaculate extension to yield 800 x 10(6) cells/mL as in P1, albeit avoiding centrifugation. Extenders A and B were milk-based and were supplemented with 5% egg yolk and fructose. Extender B was clarified by centrifugation (twice at 3310 g/20 min). Extender C was based on TRIS-citrate-fructose supplemented with 20% egg yolk and clarified as described for Extender B. Final glycerol concentration was 7% for all 3 extenders. Post-thaw parameters studied were subjective motility, computer assisted sperm motility analysis (CASA), membrane integrity (SYBR-14/P1), and capacitation status (chlortetracycline assay, CTC). The overall sperm concentration (x 10(6)/straw) differed (P<0.001) between P1 (mean+/-SD, 138.1+/-14.8) and P2 (216.5+/-13.9). Despite centrifugation, P1 appeared to be less harmful for spermatozoa than P2, yielding higher percentages of subjective motility, linearity, membrane integrity and uncapacitated spermatozoa. Due to the difference in concentrations obtained between P1 and P2, the total calculated numbers of spermatozoa having desirable characteristics were higher in samples processed as P2. In Experiment 1, P1 resulted in lower calculated numbers x 10(6) in the Aldose of subjective motility (87.2+/-5.1 vs 125.3+/-5.1; P<0.05), linearity (70.6+/-4.3 vs 79.8+/-4.3; NS), intact-membrane (77.4+/-5 vs 108.5+/-5.1; P<0.001), and uncapacitated (36.5+/-2.5 vs 46.5+/-2.5; P<0.05) spermatozoa, than P2. In Experiment 2, calculated sperm numbers (x 10(6)/straw) were lower in P1 than in P2 for subjective motility (80.8+/-5.4 vs 92.0+/-5.4; NS), linearity (63.3+/-5.6 vs 73.1+/-5.6; NS), membrane integrity (77.7+/-3.6 vs 101.0+/-3.6; P<0.001), and uncapacitated spermatozoa (28.3+/-3.24 vs. 4.1+/-3.2; P<0.01). Extender B (clarified milk extender) was consistently better than Extender A (nonclarified milk extender) for all parameters studied, but the difference was only statistically significant for linearity after 1 h of incubation at 38 degrees C (44.0+/-2.4 vs 36.2+/-2.4; P<0.05). Extender B was also better than Extender C (TRIS-citrate-fructose) for percentage of uncapacitated (49.7+/-2.2 vs 34.4+/-2.3; P<0.001), subjective motile (57.5+/-2.7 vs 43.8+/-2.7; P<0.01), and linear motile (46.5+/-2.8 vs 33.7+/-2.8; P<0.01) spermatozoa, but not for membrane integrity (51.6+/-1.5 vs 51.7+/-1.5). It was concluded that exclusion of centrifugation, as in P2, yielded higher sperm numbers with desirable characteristics per straw. Clarification of milk-based extender (B) resulted in better post-thaw sperm quality, especially compared with TRIS-based extender (C).     

Effect of solid storage at 15 degrees C on the subsequent motility and fertility of rabbit semen

We conducted two studies to improve preservation of rabbit semen. The objective of the first study was determine whether a glucose- and fructose-based extender with two different amounts of gelatin would solidify at 15 degrees C, and to evaluate the influence of gelatin supplementation on sperm motility parameters after storing semen up to 10 days at 15 degrees C. The fertility of rabbit semen diluted in the best gelatin-supplemented extender established in Study 1 and stored for up to 5 days was evaluated in the second study. In Study 1, semen was collected with an artificial vagina from 40 bucks. Each ejaculate was diluted to (80-100) x 10(6) spermatozoa/mL (1:3, semen/extender) at 37 degrees C in one of the three following glucose- and fructose-based extenders: control (standard liquid extender), semi-gel or gel (0.7 or 1.4 g gelatin in 100 mL extender, respectively). Pools of semen were allocated among 0.6 mL plastic artificial insemination (AI) guns. Thirty (10 per extender group) AI doses were immediately analyzed (0 h) and the remainder stored in a refrigerator (15 degrees C) for 12, 24, 36, 48, 72, 96, or 240 h. All doses with gelatin extenders solidified at 15 degrees C. Semen samples, prewarmed to 37 degrees C, were evaluated with a computer-assisted sperm analysis (CASA) system. The percentage of motile cells was significantly lower using the liquid compared to the gel extenders during semen storage from 0 to 96 h. Although significance was lost, these differences persisted after 240 h of storage. Motility of spermatozoa in the semi-gel extender was intermediate between that of liquid and gel extender throughout the study. Study 2 was performed on 1250 multiparous lactating does. Five homogeneous groups of 250 does previously synchronized were inseminated using semen previously stored for 120, 96, 72, 48 or 24 h, respectively. Rabbit does receiving 24 h-stored semen (diluted with the control extender used in Study 1) served as controls. The remaining females received seminal doses supplemented with 1.4 g/100mL gelatin (gel extender used in Study 1). Kindling rates for rabbit does inseminated with gelatin-supplemented (solid) semen doses stored for 48 h (88%) or 72 h (83%) were similar to those recorded for liquid controls stored for 24 h (81%), whereas rates significantly decreased when the semen was solid and stored for 96 h (64%) or 120 h (60%) before AI. In conclusion, rabbit spermatozoa were effectively stored in the solid state at 15 degrees C, with fertility preserved for up to 5 days. Solid storage of rabbit semen would facilitate commercial distribution.     

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.     

Effects of spermatozoal concentration and post-thaw dilution rate on survival after thawing of dog spermatozoa

The objectives of this study were to evaluate the effects and interactions of freezing dog semen using 4 different sperm concentrations (50 x 10(6), 100 x 10(6), 200 x 10(6) and 400 x 10(6) spermatozoa/mL) in 0.5-mL straws and diluting the thawed semen at 4 different rates (1:0, 1:1, 1:2 and 1:4) on post-thaw survival and longevity of dog spermatozoa during incubation at 38 degrees C. Fifteen ejaculates were collected from 12 dogs and pooled. The semen pool was divided into 4 aliquots containing respectively 4,200 x 10(6), 2,100 x 10(6), 1,050 x 10(6) and 525 x 10(6) spermatozoa, which were centrifuged. Sperm pellets were rediluted with TRIS-glucose-egg yolk extender containing 5% glycerol and 0.5% of Equex STM Paste to obtain the designated sperm concentrations. The semen was frozen in 0.5-mL straws 4 cm above liquid nitrogen (LN2). The straws were thawed at 70 degrees C for 8 sec and the contents of each straw were divided into 4 aliquots and diluted with TRIS buffer at 38 degrees C at rates of 1:0, 1:1, 1:2 and 1:4 (semen:buffer), respectively, making a total of 16 treatments. Sperm motility was subjectively evaluated after thawing and at 1-h intervals during 8 h of incubation at 38 degrees C. Plasma membrane integrity and acrosomal status were evaluated at 1, 3, 6, 12 and 18 h post-thaw using a triple-staining procedure and flow cytometry. For data pooled across the post-thaw dilution rate, motility was higher (P< 0.001) in samples frozen with 200 x 10(6) spermatozoa/mu. The integrity of sperm plasma membranes after 18 h incubation was higher (P<0.05) in samples frozen with 200 x 10(6) and 400 x 10(6) spermatozoa/mL. For data pooled across sperm concentration, samples diluted at a rate of 1:2 or 1:4 had better (P<0.001) motilities after 8 h of incubation than undiluted samples or those diluted at 1:1. The integrity of the sperm plasma membranes was higher (P<0.001) at increasing dilution rates. When the 16 treatments were compared, the best longevity was obtained when semen packaged at a concentration of 200 x 10(6) spermatozoa/mL was diluted immediately after thawing at 1:4 dilution rate.     

Effect of antioxidants on preservation of motility,viability and acrosomal integrity of equine spermatozoa during storage at 5 degrees C

Preservation of liquid semen at 5 degrees C is an important technique in the breeding management of horses. Oxidative damage to spermatozoa during storage is a potential cause of the decline in motility and fertility during hypothermic storage of liquid semen. The objective of this study was to evaluate the use of water-soluble and lipid-soluble antioxidants to improve the maintenance of motility of equine spermatozoa at 5 degrees C during storage for 72 to 96 h. In Experiment 1, the effect of addition of catalase on the maintenance of motility, viability and acrosomal integrity was determined. Semen was collected, and these treatments were applied: catalase (0, 100 or 200 U/mL) in nonfat, dried skim milk extender (NFDSM; with or without seminal plasma) or 10% seminal plasma + NFDSM. Motility was determined by computerized semen analysis (CASA) at 0, 24, 48 and 72 h. Viability and acrosomal integrity were determined at 72 h of storage. There was no significant treatment effect on the maintenance of sperm motility during 72 h storage. In Experiment 2, the effect of adding lipid-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted to a final concentration of 25 x 10(6) sperm/mL in NFDSM containing butylated hydroxytoluene (BHT; 2.0, 1.0, or 0.5 mM), Vitamin E (4.0, 2.0, 1.0 mM), or Tempo (2.0, 1.0, or 0.5 mM). Although the addition of BHT significantly reduced (P < 0.05) progressive motility during storage compared to the control, there were no positive treatment effects of either Vitamin E or Tempo on maintenance of motility. In Experiment 3, the effect of adding water-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted in NFDSM containing these treatments: Trolox (2.0 mM), Tempo (1.0 mM), Vitamin C (0.45 mg/mL), BSA (3% w/v), combinations of these antioxidants, or control. Adding these water-soluble antioxidants did not significantly improve the maintenance of motility during cooled storage at 5 degrees C. In conclusion, adding the enzyme scavenger, catalase, or a variety of lipid- and water-soluble antioxidants did not significantly improve the maintenance of motility during liquid semen storage at 5 degrees C.     

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.     

Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin

The process of cryopreservation impairs sperm cell function, potentially leading to a reduction in fertility. The objectives of the present study were to evaluate the effects that cryopreservation using two different extenders has on sperm motility and mitochondrial function, as well as on the integrity of plasma membranes, acrosomal membranes and chromatin, using practical and objective techniques. The focus of the present study was to identify correlations between alterations in sperm membranes and sperm motility in cryopreserved bovine spermatozoa. Seven ejaculates were collected from eight Simmental bulls (n=56). After collection, semen volume and concentration were assessed for purposes of dilution. Sperm motility was evaluated subjectively and by computer-assisted semen analysis, morphological characteristics were evaluated by differential interference microscopy, the integrity of plasma and acrosomal membranes, as well as mitochondrial function, were determined using a combination of fluorescent probes containing fluorescein isothiocyanate-Pisum sativum agglutinin, propidium iodide or 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. Chromatin integrity was evaluated using the acridine orange technique. The semen was subsequently divided into two aliquots and diluted with one of two extenders (Bioxcell or Botu-Bov), after which both were packaged in 0.5 mL straws and frozen using an automated system. Two straws of semen from each treatment were thawed, and the semen parameters were evaluated as described above. Cryopreservation of sperm reduced motility, damaging plasma and acrosomal membranes, as well as decreasing mitochondrial function. The Botu-Bov extender was more effective in preserving sperm motility and membrane integrity than was the Bioxcell extender.     

Effect of seminal plasma on the cryopreservation of equine spermatozoa

Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80% seminal plasma for 15 min, prior to being diluted to a final concentration of 50 x 10(6)sperm/mL in a Lactose-EDTA freezing diluent and cryopreserved. Sperm motility was analyzed at 10 and 90 min after thawing, while sperm viability and acrosomal integrity were analyzed 20 min after thawing. Seminal plasma did not affect sperm motility, viability or acrosomal integrity (P>0.05). Experiment 2 tested the main affects of seminal plasma level (5 or 20%), incubation temperature (5 or 20 degrees C) and incubation time (2, 4 or 6 h) prior to cryopreservation. In this experiment, spermatozoa were incubated with 5 or 20% seminal plasma for up to 6h at either 5 or 20 degrees C prior to cryopreservation in a skim milk, egg yolk freezing extender. Samples cooled immediately to 5 degrees C, prior to freezing had higher percentages of progressively motile spermatozoa than treatments incubated at 20 degrees C (31 versus 25%, respectively; P<0.05), when analyzed 10 min after thawing. At 90 min post-thaw, total motility was higher for samples incubated at 5 degrees C (42%) compared to 20 degrees C (35%; P<0.05). In addition, samples containing 5% seminal plasma had higher percentages of total and progressively motile spermatozoa (45 and 15%) than samples exposed to 20% seminal plasma (33 and 9%; P<0.05). In conclusion, although the short-term exposure of sperm to seminal plasma had no significant effect on the motility of cryopreserved equine spermatozoa, prolonged exposure to seminal plasma, prior to cryopreservation, was deleterious.     

Sperm concentration at freezing affects post-thaw quality and fertility of ram semen

We have investigated the effect of sperm concentration in the freezing doses 200, 400, 800, and 1600 × 10⁶ mL⁻¹ on the post-thaw quality and fertility of ram semen. Semen was collected from seven adult Churra rams by artificial vagina during the breeding season. The semen was diluted in an extender (TES-Tris-fructose, 20% egg yolk, and 4% glycerol), to a final concentration of 200, 400, 800, or 1600 × 10⁶ mL⁻¹ and frozen. Doses were analyzed post-thawing for motility (computer-assisted sperm analysis system [CASA]), viability, and acrosomal status (fluorescence probes propidium iodide [PI]/peanut agglutinin conjugated with fluorescein thiocyanate (PNA-FITC), SYBR-14/PI [Invitrogen; Barcelona, Spain] and YO-PRO-1/PI [Invitrogen; Barcelona, Spain]). Total motility and velocity were lower for 1600 × 10⁶ mL⁻¹ doses, while progressive motility and viability were lower both for 800 and 1600 × 10⁶ mL⁻¹. The proportion of viable spermatozoa showing increased membrane permeability (YO-PRO-1+) rose in 800 and 1200 × 10⁶ mL⁻¹. Intrauterine inseminations were performed with the 200, 400, and 800 × 10⁶ mL⁻¹ doses at a fixed sperm number (25 × 10⁶ per uterine horn) in synchronized ewes. Fertility (lambing rate) was similar for semen frozen at 200 (57.5%) or 400 × 10⁶ mL⁻¹ (54.4%), whereas it was significantly lower for 800 × 10⁶ mL⁻¹ (45.5%). In conclusion, increasing sperm concentration in cryopreserved semen, at least at 800 × 10⁶ mL⁻¹ and more, adversely affects the postthawing quality and fertility of ram semen.     

Cryopreservation of goat spermatozoa: comparison of two freezing extenders based on post-thaw sperm quality and fertility rates after artificial insemination

TRIS-glucose or skim milk extenders are most commonly used for cryopreserving goat sperm. The aim of this study was to compare the ability of two extenders based on TRIS and skimmed milk buffer to maintain sperm viability after cryopreservation. Goat semen samples (n=110) were frozen with TRIS and with milk extender and thaw. Sperm motion parameters, morphology and acrosomal integrity were assessed in fresh and frozen-thawed samples by Sperm Class Analyzer (SCA) and Diff-Quik and Spermac staining techniques. Pregnancy rates were obtained after cervical insemination with frozen semen doses. The cryopreservation process had a significant effect on acrosome and kinematic parameters. TRIS extender provided more effective preservation of total motility, velocity parameters and amplitude of lateral head displacement after freezing. The percentage of acrosome intact spermatozoa was significantly higher in samples diluted with milk extender. In the insemination doses, mean values of velocity parameters and lateral head displacement were higher in doses processed in TRIS. Spermatozoa frozen in milk extender was mathematically greater than for those frozen with TRIS extenders, though no significant difference exists. We conclude that post-thaw kinematic parameters and acrosome integrity assessed after 1h of incubation was acceptable in both extenders which indicated the feasibility of cryopreserving goat spermatozoa. TRIS extender results in better in vitro performance compared to milk, though these improvements were not reflected in fertility results. Semen doses cryopreserved in milk extender provided greater pregnancy rates after intra-cervical insemination compared to those in TRIS extender (52.4% versus 42.9%).     

Influence of centrifugation or low extension rates prefreezing on the fertility of ram semen after cervical insemination

We compared the fertility of thawed ram semen, frozen according to different prefreezing semen handling protocols and previously well-defined in vitro, after cervical artificial insemination (AI) during natural estrus in Corriedale sheep. Following primary extension 1 + 1, we adjusted the final sperm concentration before packaging (200 x 10(6)/straw) either by centrifugation, in order to reconcentrate the extended semen (Protocol 1: P1), or without centrifugation, by adjusting the final sperm number by stepwise extension (Protocol 2: P2). We evaluated sperm motility (assessed both subjectively and with a computer-assisted sperm analysis instrument [CASA]), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline [CTC]) in vitro in three pooled straws of frozen-thawed semen. Three hundred Corriedale ewes, having shown spontaneous estrus during the breeding season (i.e., April, in the southern hemisphere) under extensive management conditions in Uruguay, were cervically inseminated with thawed semen from the same freezing operations as studied in vitro. The semen evaluation in vitro yielded higher percentages (P < 0.05) of damaged spermatozoa in the samples where sperm numbers were adjusted by extension before freezing (P2), compared with when adjustment was done by centrifugation (P1). However, due to the higher sperm concentration finally achieved by P2, the calculated total number of viable spermatozoa was almost equal in the two AI doses. We observed no differences in fertility between P1 and P2 for either nonreturn rates (NRRs) 21 (30.8 vs. 29.7%) and 36 (28.5 vs. 27.8%) days after AI or lambing rate (21.9 vs. 21.4%), respectively. Fertility did not differ significantly between the two different procedures of adjusting sperm numbers prior to freezing. This may indicate that the simplified protocol with adjusted extension of the semen, resulting in higher numbers of viable spermatozoa, should be the procedure of choice when freezing ram semen under field conditions. Further studies aimed at improving the modified protocol need to be performed.     

Activity of Chinchilla laniger spermatozoa collected by electroejaculation and cryopreserved

Because reproductive studies and the application of assisted reproductive techniques are relevant issues for an endangered species such as Chinchila laniger, the availability of a source of viable spermatozoa becomes of utmost importance. In this paper, we evaluate several functional parameters (motility, viability, response to hypoosmotic swelling test and acrosomal integrity) of fresh or frozen-thawed spermatozoa. Electro-ejaculation trials (50-cyc/sec sinusoidal wave was applied for 5 of every 10 sec) were successful in all unanesthetized animals. After volume (108.3 +/- 12.0 microL, n = 15) and concentration (421.8 +/- 34.4 x 10(6) cells/mL, n = 15) measurements, the above mentioned parameters were determined. In frozen-thawed semen samples sperm motility, viability, hypoosmotic swelling test and acrosomal integrity were significantly lower than in fresh semen samples. The results clearly indicated that electro-ejaculation is a useful method for evaluating spermatozoa for genetic analysis or for used in Al in this species. In addition, the cryopreservation procedure in this study preserved adequate levels of functional sperm activity.     

A comparison of BoviPure and Percoll on bull sperm separation protocols for IVF

In the present study, we have examined the effect of density gradient preparations BoviPure and Percoll on bull sperm separation and the in vitro fertilization (IVF) and culture (IVC) results. Frozen/thawed semen from five simmental bulls were pooled. Sperm quality parameters such as sperm motility, concentration, membrane activity (HOS assay), membrane integrity (SYBR-14/PI assay) and acrosomal status (EthD-1/FITC-PSA assay) were evaluated before and after sperm processing for IVF using BoviPure and Percoll density gradient separations. The results of the evaluated parameters before sperm processing were: motility 50%, concentration 82.33x10(6)spz/mL, membrane activity 39.05%, membrane integrity 42.97% and the acrosomal status 46.90% of the live spermatozoa with intact acrosomes. After sperm processing with BoviPure and Percoll the motility was 66.67 and 64.17%, the concentration was 25.50x10(6) and 27.67x10(6)spz/mL, the membrane activity was 53.78 and 56.58%, the membrane integrity was 70.85 and 68.76% of and the acrosomal status was 74.16 and 67.46% of the live spermatozoa with intact acrosomes, respectively. Percentages were referred to the total number of spermatozoa. There were significant differences (P<0.05) between the evaluated parameters before and after sperm processing for both separation protocols. We found no significant differences (P>0.05) regarding sperm evaluation parameters between the protocols. A total of 492 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA in six replicates. The cleavage (D2) and blastocysts (D7) rate were significantly higher (P<0.05) for the BoviPure group compared to the Percoll group: 75.80 and 28.21%; 61.58 and 20.83%, respectively. However, the number of hatched blastocysts (D10) did not differ significantly between sperm separation protocols (P>0.05). Our results indicate that both protocols gave suitable sperm for IVF. This finding and the similarity between those two density gradient preparations suggests that BoviPure is a good alternative for sperm separation in bovine IVF.     

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.     

Comparison of two dilution rates on canine semen quality after cryopreservation in a coconut water extender

The objective of the present study was to evaluate the effect of sperm dilution (one part semen:one part extender or at 200 x 10(6) spermatozoa/mL) using a coconut water extender on the post-thaw sperm quality. Twelve ejaculates were collected from six dogs. Semen was divided into two aliquots, one for dilution one part semen:one part extender (group 1) and another for a concentration of 200 x 10(6) spermatozoa/mL (group 2). Semen was initially extended at 37 degrees C at a proportion of one part semen:half part extender (1:1/2) for group 1 (A-fraction). For group 2, the volume for a concentration of 200 x 10(6) spermatozoa/mL was calculated and a half of this volume was used for the initial dilution (A-fraction, 37 degrees C). Coconut water extender containing 20% egg yolk was used for this initial dilution in both groups. After dilution, the semen was cooled for 40 min in a thermal box (15 degrees C) and for 30 min in a refrigerator. The other half of the extender (B-fraction) containing egg yolk and glycerol (12%) was added to semen in both groups. Subsequently, the final concentration of glycerol in the extender was 6%. Ejaculates were frozen in 0.25 mL straws 5 cm above the surface of liquid nitrogen and stored at -196 degrees C. After 1 week, straws were thawed at 37 degrees C for 1 min and the microscopic criteria were evaluated. The dilution method had no influence on sperm motility, vigor and normal spermatozoa (71.4 compared with 67.7%). There was no effect of dog, ejaculate within male on post-thaw semen quality. Moreover, there was not a male x treatment interaction. Both treatments were efficient in preserving sperm quality.     

The effect of different extenders on post-thaw sperm survival, acrosomal integrity and longevity in cryopreserved semen of Formosan Sika deer and Formosan Sambar deer

This study investigates the efficacy of five extenders in contributing to the outcome of semen cryopreservation in Formosan Sika and Sambar deer. Pooled semen (n=4) of six males of each breed was used. In Sika deer, semen collection rate was 96% (23/24) over all electro-ejaculations. Volume, sperm motility and sperm concentration of fresh ejaculates was 0.5+/-0.4 ml, 77+/-6% and 1471.3+/-940.0 x 10(6) ml(-1), respectively. Post-thaw motility in respective extender was A: 66+/-16%; B: 71+/-2%; C: 73+/-6%; D: 9+/-4% and E: 26+/-12% (mean+/-S.D.). In extender C (74+/-14%) more viable spermatozoa were preserved than in the others (A: 64+/-10%; B: 48+/-11%; D: 41+/-16%; E: 47+/-6%; P<0.05). Acrosomal integrity was not influenced by extender composition. Post-thaw motility did not decrease during a 4-h incubation period, irrespective of the extender used (P>0.05). In Sambar deer, semen collection rate was 88% (21/24) over all electro-ejaculations. Volume, sperm motility and sperm concentration of fresh ejaculates was 1.3+/-0.5 ml, 82+/-4% and 379.1+/-252.2 x 10(6) ml(-1), respectively. Post-thaw motility was in respective extenders A: 69+/-2%; B: 74+/-6%; C: 73+/-2%; D: 13+/-6% and E: 31+/-20%. Extenders B and C were superior (P>0.05) with respect to sperm motility. Similarly, post-thaw viability in extenders A (70+/-7%), B (76+/-7%) and C (79+/-2%) was higher than that D (25+/-19%) and E (29+/-17%) (P<0.01). Sperm acrosomal integrity was better preserved in extenders B (86+/-4%) and C (83+/-4%) than in extenders A (54+/-13%), D (39+/-22%) and E (46+/-22%) (P<0.05). Post-thaw sperm longevity in extender A reduced from 69 to 16% during incubation (P<0.05) whereas only a slight decrease was observed in the other extenders after 4 h. In conclusion these data show that egg-yolk-Tris-Tes-glycerol based extender C containing Equex STM paste is optimal for freezing semen of Formosan Sika deer while egg-yolk-Tris-citric acid-glycerol based extender B containing Equex and extender C are superior in semen cryopreservation to others for Formosan Sambar deer.     

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.