Effect of different number of frozen spermatozoa inseminated on the reproductive performance of rabbit does

The relationship between the number of frozen spermatozoa inseminated and fertility rate and litter size at birth in rabbit does was investigated. Six hundred artificial inseminations (AI) were performed on multiparous lactating does with three spermatozoa concentrations: 10, 25, 50x10(6)spermatozoa/AI. All the does were synchronized with 20 UI of eCG 2 days before AI. The estimated sexual receptivity was 87%. The freezing-thawing procedure strongly reduced kinetic and functional traits (acrosome integrity, capacitation) of the spermatozoa. The number of spermatozoa inseminated did not affect the reproductive performance: the mean fertility rate and litter size values were 51.5% and 7.6%, respectively. Sexually receptive does (n=522) inseminated with frozen spermatozoa showed a 58.0% fertility rate whereas, non-receptive does (n=78), had a very poor fertility rate (7.8%).     

The relationship between rabbit semen characteristics and reproductive performance after artificial insemination

The relationships between several rabbit buck semen traits, concerning either the ejaculate or the dose inseminated (volume, mass motility, pH, percentage of motile sperm (PMS), concentration, number of total and motile sperm per ejaculate and per insemination dose) and the reproductive performance of does was investigated in 839 inseminations involving 54 bucks and 111 does. Four genetic types were involved: INRA1601 strain (A), INRA2066 strain (B) and the two reciprocal crossbreds (AB and BA). The mating design was A x A, B x B, (AB or BA) x (AB or BA). Semen was diluted (1:9) and a constant volume of 0.5 ml was inseminated 2-4h after collection. Therefore, the total number of spermatozoa per dose was variable and proportional to the initial concentration. Mass motility significantly influenced the kindling rate. Taken separately, volume, PMS and concentration did not influence the kindling rate but their product, the number of motile sperm per ejaculate, did. Litter size (total born) was significantly influenced by concentration and all variables depending on it, particularly the number of total and motile sperm per dose. However, reproductive performances were predominantly influenced by the physiological status of the does at insemination (lactation stage and receptivity).     

Influence of supplemental dietary sprouted wheat on reproduction in artificially inseminated doe rabbits

The sprouted wheat contains great amounts of 6-methoxybenzoxazolinone (6-MBOA) a phenol compound that stimulates reproduction in certain small wild herbivorous mammals. The objective of the present study was to evaluate the effect of dietary sprouted wheat as supplement on reproduction in artificially inseminated doe rabbits. Two-month-old New Zealand White doe rabbits (n = 48) were randomly allocated to one of two treatments: (1) pelleted diet plus sprouted wheat for 6 consecutive days prior to service, (2) pelleted diet only (control). First insemination occurred when does reach 3200g body weight and bred 6 days after parturition across 6 months period during summer and autumn. Records from 41 does completing the experiment accounted for 192 inseminations and 142 kindlings equivalent to 4.6 +/- 0.15 and 3.5 +/- 0.15 litters per doe, respectively. Mean daily intake of sprouted wheat on wet and dry matter bases were 196.1 +/- 7.5 and 75.4 +/- 2.9g, respectively. The number of young born produced per doe during the trail was significantly (P = 0.02) greater in does fed sprouted wheat (28.1 +/- 1.2 versus 23.6 +/ -1.3 control). Does fed sprouted wheat had 0.65 +/- 0.06 receptivity rate at AI over 28 per cent greater (P = 0.001) than does in the control treatment. Sexual receptivity was not influenced by physiological status of does and season (P > 0.05). Diet and season had no effect (P>0.05) on kindling rate. However, physiological status had an effect (P = 0.002) on this variable. Kindling rates for nulliparous, lactating and non-lactating does were 0.95 +/- 0.08, 0.63 +/- 0.04 and 0.78 +/ -0.11, respectively. Sexually receptive does had a greater (P = 0.0001) kindling rate (0.95 +/- 0.05) than non-receptive females (0.63 +/-0.05). Does fed sprouted wheat produced larger (P = 0.02) litters than those in the control group: 7.7 +/- 0.30 and 6.8 +/- 0.32, respectively. There was an effect of season (P = 0.04) on the size of the litter at birth. Largest litters were born during autumn (7.9 +/- 0.37) than during summer (6.6 +/- 0.41). Receptive does had more (P = 0.002) young in the litter (7.9 +/- 0.28) than non-receptive (6.6 +/- 0.35). Feeding sprouted wheat as a source of biological 6-MBOA enhanced sexual receptivity and prolificacy in artificially inseminated doe rabbits bred in summer and autumn.     

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.     

Effect of sperm number and frequency of insemination on fertility of mares inseminated with cooled semen

In this study, we tested the hypothesis that insemination of mares with twice the recommended dose of cooled semen (2 x 10(9) spermatozoa) would result in higher pregnancy rates than insemination with a single dose (1 x 10(9) spermatozoa) or with 1 x 10(9) spermatozoa on each of 2 consecutive days. A total of 83 cycles from 61 mares was used. Mares were randomly assigned to 1 of 3 treatment groups when a 40-mm follicle was detected by palpation and ultrasonography. Mares in Group 1 were inseminated with 1 x 10(9) progressively motile spermatozoa that had been cooled in a passive cooling unit to 5 degrees C and stored for 24 h. A second aliquot of semen from the same collection was stored for an additional 24 h and inseminated at 48 h after collection. Mares in Group 2 were inseminated once with 1 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. Group 3 mares were inseminated once with 2 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. All mares were given 2500 IU i.v. hCG at the first insemination. Pregnancy was determined by ultrasonography 12, 14 and 16 d after ovulation. On Day 16, mares were administered i.m. 10 mg of PGF2 alpha and, upon returning to estrus, were randomly reassigned to a group for repeated treatment. Semen was collected from one of 3 stallions every 3 d; mares with a 40-mm ovarian follicle were inseminated with semen from the stallion collected on the preceding day. Semen was allocated into doses containing 1 x 10(9) progressively motile spermatozoa, diluted with dried skim milk-glucose extender to a concentration of 25 x 10(6) motile spermatozoa/ml (total volume 40 ml), placed in a passive cooling unit and cooled to 5 degrees C for 24 or 48 h. Response was measured by number of mares showing pregnancy. Data were analyzed by Chi square. Mares inseminated twice with 1 x 10(9) progressively motile spermatozoa on each of two consecutive days had a higher pregnancy rate (16/25, 64%; P < 0.05) than mares inseminated once with 1 x 10(9) progressively motile spermatozoa (9/29, 31%) or those inseminated once with 2 x 10(9) progressively motile spermatozoa (12/29, 41%). Pregnancy rates did not differ significantly (P > 0.10) among stallions (69, 34 and 32%). Interval from last insemination to ovulation was 0.9, 2.0 and 2.0 d for mares in Groups 1, 2 and 3, respectively. Based on these results, the optimal insemination regimen is a dose of 1 x 10(9) progressively motile spermatozoa given on two consecutive days. However, a shorter interval (< or = 24 h rather than > 0.9 d) between insemination and ovulation may affect pregnancy rates, and needs to be investigated.     

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.     

Fertility of weaned sows after deep intrauterine insemination with a reduced number of frozen-thawed spermatozoa

The present study evaluates the effectiveness of the transcervical deep intrauterine insemination (DUI) with a reduced number of frozen-thawed boar spermatozoa in weaned sows. DUI was performed using a specially designed flexible device (length 180 cm, outer diameter 4mm, working channel 1.8mm, working channel's volume 1.5 ml) that was inserted through an artificial insemination spirette to cross the cervix lumen and moved into one uterine horn as far as possible. Spermatozoa diluted in 7.5 ml of BTS were flushed into the uterine horn by a syringe attached to the working channel. In Experiment 1, 111 hormonally treated (eCG/hCG) weaned sows were inseminated once using one of the following three regimens: (1) DUI with frozen-thawed spermatozoa (1000 x 10(6) cells per dose; n=49); (2) DUI with fresh semen (150 x 10(6) cells per dose; n=29, as control of DUI procedure); and (3) cervical insemination with frozen-thawed spermatozoa (6000 x 10(6) cells diluted in 100ml; n=33). No differences (P>0.05) were found for farrowing rates (77.55, 82.76, and 75.76, respectively) or litter sizes (9.31+/-0.41, 9.96+/-0.32, and 9.60+/-0.53 piglets born per litter, respectively) among the groups. In Experiment 2, DUI was performed on the spontaneous estrus in weaned sows (2-6 parity) with 1000 x 10(6) frozen-thawed (40 sows) or 150 x 10(6) fresh spermatozoa (38 sows). The farrowing rate of sows inseminated twice with frozen-thawed spermatozoa (70%) was significantly (P<0.05) lower than with fresh semen (84.21%). No significant difference (P>0.05) was found in litter size between frozen-thawed spermatozoa (9.25+/-0.23 piglets born per litter) and fresh semen (9.88+/-0.21 piglets born per litter). These preliminary results indicate that application of DUI provides acceptable fertility in weaned sows using a relatively low number of frozen-thawed spermatozoa.     

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.     

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.     

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 ).     

Birth of offspring of pre-determined sex after artificial insemination of frozen-thawed, sex-sorted and re-frozen-thawed ram spermatozoa

The fertility of ram spermatozoa cryopreserved prior to, and following, sex-sorting by flow cytometry was assessed after insemination of mature Merino ewes at a synchronised estrus. Ewes were inseminated with spermatozoa from three rams, split into four treatment groups: 50 x 10(6) motile non-sorted, frozen-thawed (Control50), 15 x 10(6) motile non-sorted, frozen-thawed (Control15), 15 x 10(6) motile sex-sorted, frozen-thawed (SF15) or 15 x 10(6) motile frozen-thawed, sex-sorted, re-frozen-thawed (FSF15) ram spermatozoa. Separation of SF15 and FSF15 treatments into X- and Y-chromosome-bearing populations was achieved using a high-speed sperm sorter. The percentage of ewes lambing after insemination was similar for Control15 (36/74; 48.6%), SF15 (35/76; 46.1%) and FSF15 (26/72; 36.1%) groups (P>0.05). A higher percentage of ewes produced lambs in the Control50 (38/70; 54.3%) than the FSF15 group (P<0.05). Fifty-one of the 55 (92.7%) lambs derived from fresh, sex-sorted frozen-thawed spermatozoa were of the predicted sex, as were 41/43 (95.3%) lambs derived from frozen-thawed, sex-sorted, re-frozen-thawed spermatozoa. This study demonstrated for the first time in any species that frozen-thawed spermatozoa, after sex-sorting and a second cryopreservation step, are capable of producing offspring of the predicted sex following artificial insemination.     

Comparison of pregnancy outcome in mares among methods used to evaluate and select spermatozoa for insemination.

An artificial insemination dose for mares consisting of 500 million progressively motile spermatozoa is considered "standard" by most clinicians. However, little information is available directly comparing pregnancy outcome among methods of evaluating and selecting spermatozoa for insemination. The objective of this study was to determine if the method of spermatozoal evaluation and selection influences fertility as measured by pregnancy outcome. Mares were inseminated with 100 or 500 million spermatozoa that were selected for progressive motility, normal morphology, hypoosmotic swelling or absolute number regardless for evaluation method or quality. Thirty-two breeding cycles were tested for each treatment group and at each spermatozoal dose. Pregnancy outcomes were 44 and 41%, 55 and 41%, 39 and 31%, and 45 and 41%, for the 100 and 500 million progressively motile, morphologically normal, hypoosmotic swelling positive and absolute number treatment groups, respectively. Pregnancy outcome did not differ among methods of spermatozoal evaluation and selection for artificial insemination in the 100 (P=0.52) or 500 (P=0.78) million spermatozoa groups. Also the total number of spermatozoa and the absolute number of progressively motile, morphologically normal or hypoosmotic swelling positive spermatozoa inseminated, were not closely associated with pregnancy outcome in the 100 (P=0.24, 0.29, 0.33 and 0.38, respectively) or 500 (P=0.20, 0.84, 0.50 and 0.74, respectively) million spermatozoa groups. In this study, we found that the method of spermatozoal evaluation did not offer an advantage for pregnancy when used to select spermatozoa for insemination at the doses tested. These results were surprising, as we expected there would be differences among the evaluation methods. Instead, we found that evaluating spermatozoa offered no advantage for pregnancy over simply inseminating with a specified number of spermatozoa not selected for any particular characteristic under the conditions of our experiment.     

Detrimental effect on availability of buserelin acetate administered in seminal doses in rabbits

The study evaluated a seminal effect on the ability to induce ovulation of a synthetic GnRH analogue, buserelin acetate, administered by vaginal mucosa in rabbit does. In a first experiment, 751 receptive nulliparous and multiparous non-lactating does were randomly assigned to groups of different seminal doses (6, 12, 24, 50, and 100 million total sperm in 0.5 mL). All seminal doses contained 5μg of buserelin acetate to induce ovulation by vaginal mucosa absorption. Two hundred and six does from 751 were laparoscopized at 12^th days of gestation to evaluate ovulation induction, ovulation rate and implanted embryos, while pregnancy rate and total and live born were noted in all females. Results showed that the pregnancy rate was significantly affected by the seminal dose used (0.82 vs 0.72, 0.50, and 0.45, for 6, 24, 50, and 100 million of spermatozoa, respectively). Data from laparoscopized does showed significant differences between the group of 6 and 50 million sperm dose in the ovulation induction and consequently in the pregnancy rate (0.79 vs 0.52, 0.79 vs 0.48, respectively). Does from all groups had similar implanted embryos and litter sizes irrespective of seminal dose used. In a second experiment, inseminations were done without spermatozoa, 0.5 mL of two dilutions of seminal plasma (1/4 and 1/20) with 5μg of buserelin acetate were introduced into vagina from 71 receptive females and its results were compared to a control group (35 does) induced to ovulate with 1μg of buserelin acetate administered intramuscularly. Only 40% of females from 1/4 plasma dilution group became to ovulate. Consequently, the dilution rate of seminal plasma may reduce the availability rate of the GnRH analogue and the concentration needed to provoke the ovulation induction.     

Sources of variation in the reproductive performance of ewes inseminated with frozen-thawed ram semen by laparoscopy

Fertility data from 8 artificial insemination programs, involving more than 5000 ewes and 110 rams in 3 flocks, were analyzed to determine variation due to individual AI program and ram in the reproductive performance of ewes inseminated with frozen-thawed semen by laparoscopy. The semen had been previously frozen by commercial AI centers in either pellets or straws. Both AI program and individual ram affected the proportion of ewes pregnant and the number of fetuses per ewe inseminated, but not the number of fetuses per pregnant ewe. Semen samples from 97 of the rams used were analyzed on a Hamilton Thorn HTM 2000 image analyzer for sperm concentration, percentage of motile and progressively motile spermatozoa, mean progressive velocity, and mean linear index. The correlations between these traits and reproductive performance obtained after insemination were calculated. There was large variation in the quantity and quality of the frozen semen, but only the number of total and motile spermatozoa inseminated per ewe was correlated with fertility (0.25 and 0.26, respectively). Regression analysis showed that none of the traits measured were useful for predicting fertility.     

Effect of insemination dose on pregnancy rate in mares

Different insemination doses have been used for artificial insemination(AI) in horses. Since the insemination dose can affect the pregnancy rate, it is important to ensure that an adequate dose be used regardless of the type of inseminationprotocol used. The aim of this study was to find out if it is possible to decrease the insemination dose from 500 x 10(6) progressively motile spermatozoa to 300 x 10(6) progressively motile spermatozoa and still maintain an acceptable pregnancy rate when using extended fresh semen. Thirteen stallions of known fertility and a well-defined group of 64 mares were used in the study. The mares were randomly assigned to 1 of 2 insemination groups. Examination for pregnancy was performed by ultrasonography per rectum approximately 16 d after the last insemination. When using an insemination dose of 300 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 75%. With an insemination dose of 500 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 64%. There was no significant difference in the pregnancy rate between the 2 insemination doses (P = 0.341). We conclude that when using fresh extended semen it is unlikely that an insemination dose of 300 x 10(6) progressively motile spermatozoa would yield a lower pregnancy rate than a dose of 500 x 10(6) progressively motile spermatozoa if stallions with good quality semen are selected.     

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.     

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.     

Homologous prostatic fluid added to frozen-thawed dog spermatozoa prior to intravaginal insemination of bitches resulted in better fertility than albumin-free TALP

The aim of this study was to determine whether canine prostatic fluid has intrinsic effects resulting in higher fertility than albumin-free Tyrode's albumin lactate pyruvate (afTALP) when added to thawed semen prior to intravaginal insemination. Twenty-four German shepherd bitches were inseminated intravaginally with frozen-thawed spermatozoa to which either homologous prostatic fluid (Group P; 12 bitches) or afTALP (Group T; 12 bitches) was added to give a final insemination volume of 7mL. Each bitch was inseminated daily starting when the vaginal folds first became angular and continuing until the day before a diestrus vaginal smear was first seen. Bitches were spayed about 3 weeks after the onset of diestrus and the number of corpora lutea and the number of conceptuses counted. Group P and Group T bitches were, respectively, inseminated 5.3+/-1.0 and 5.8+/-2.1 times with 48.9+/-8.6 and 50.4+/-8.3 million progressively motile spermatozoa per insemination. Eight Group P bitches and 10 Group T bitches conceived with totals of 76 and 45 conceptuses and 126 and 117 corpora lutea, respectively. Odds of conception were taken as the number of conceptuses divided by (the number of corpora lutea minus the number of conceptuses). After adjustment for the number of progressively motile spermatozoa per day and the random effect of bitch, the addition of prostatic fluid resulted in an increased odds of conception compared to afTALP. This effect decreased as the number of progressively motile spermatozoa per day increased.     

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.     

Hysteroscopic insemination of small numbers of spermatozoa at the uterotubal junction of preovulatory mares

Mares were inseminated with motile spermatozoa suspended in 30-150 microliters Tyrode's medium directly onto the uterotubal papilla at the anterior tip of the uterine horn, ipsilateral to the ovary containing a dominant preovulatory follicle of > or = 35 mm in diameter, by means of a fine gamete intrafallopian transfer (GIFT) catheter passed through the working channel of a strobed light videoendoscope. Insemination of 10, 8, 25, 14, 11 and 10 mares with, respectively, 10.0, 5.0, 1.0, 0.5, 0.1 or 0.001 x 10(6) motile spermatozoa resulted in conception rates of, respectively, 60, 75, 64, 29, 22 and 10%. Deposition of 1.0 x 10(6) motile spermatozoa onto the uterotubal papilla began to approach the limit of successful fertilization. These doses are far lower than the 3-15 x 10(9) spermatozoa normally ejaculated by fertile stallions during mating, and the accepted minimum dose of 500 x 10(6) spermatozoa used for conventional uterine body insemination in mares. The simplicity of the technique offers a practical means of exploiting new breeding technologies that require very small numbers of spermatozoa in horse breeding.