Unilateral intrauterine horn insemination of fresh semen in cats

The sperm count required were investigated to obtain a conception rate of 80% by unilateral intrauterine insemination (UIUI) of fresh semen in cats. The conception rates obtained by insemination before and after ovulation were also examined. Thirty-six female cats aged 1-7 years were used in the experiments, and the number of experimental cases was 44. Seven male cats aged 2-12 years from which semen could be collected by the artificial vagina method were used. In artificial insemination, 100 iu x 2 or 250 iu of hCG was administered on days 2-4 of estrus, and sperm were introduced into the uterine horn with a greater number of ovulations (or mature follicles) 15, 20 and 30 hr after hCG administration by laparotomy. The inseminated sperm counts were 2 x 10(6) (Exp. 1). 4 x 10(6) (Exp. 2), and 8 x 10(6) (Exp. 3). As a result, ovulation was induced in 42 of 44 cases (induction rate: 95.5%) regardless of the dosage of hCG. Conception was obtained by UIUI in two of 16 animals (conception rate: 12.5%) in the Exp. 1, five of 16 animals (31.3%) in Exp. 2, and eight of 10 animals (80.0%) in Exp. 3. Regarding the relationship between the ovulation state at insemination and conception, the conception rate obtained by insemination before ovulation was clearly higher than that obtained by insemination after ovulation (p<0.05). Regarding the number of kits compared to the number of ovulations on the inseminated side, the percentages of cases in which the number of kits exceeded the number of ovulations on the inseminated side were similar in all groups inseminated with a different number of sperm. It is therefore necessary to investigate conception rates obtained by bilateral insemination to increase the fertility rate. Based on the above findings, it was shown that the sperm count required for fertilization by UIUI is 8 x 10(6).     

Unilateral intrauterine horn insemination of frozen semen in cats

Frozen feline semen was prepared using two types of extenders, egg yolk Tris-fructose citric acid (EYT-FC) and egg yolk sodium citrate solution (EYC), and the semen qualities after thawing and the conception rates obtained by unilateral intrauterine horn insemination (UIUI) were investigated. Cats used in the experiment were six males and 11 females aged 2-12 years (the number of experimental cases was 17). For preparation of frozen semen, semen collected by the artificial vagina method was adjusted to I x 10(8) sperm/m/ and 7% glycerol, put in 250 microl straws, and then frozen using a cell freezer. The mean sperm motility after thawing was 30.0+/-9.7 (SE) % in the semen prepared with EYT-FC and 30.0+/-3.3% in the semen prepared with EYC. Four of seven animals were fertilized by UIUI using two straws in both extenders, and the conception rate was 57.1%. The mean ratios of number of kits to the number of ovulations in the inseminated side were 61.1+/-24.5% and 30.5+/-3.4% for EYT-FC and EYC, respectively, showing that the ratio tended to be higher in the semen prepared with EYT-FC. The above findings, comparing the two extenders for preparation of frozen feline semen, showed that EYT-FC is slightly superior to EYC. To increase conception and fertility rates, it may be important to increase the sperm count for insemination and to inseminate both uterine horns.     

Intrauterine and intravaginal insemination with frozen canine semen using an extender consisting of orvus ES paste-supplemented egg yolk tris-fructose citrate

Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.     

Determination of optimal time for mating by artificial insemination with chilled semen using luteinizing hormone surge as an indicator in beagles.

Artificial insemination (AI) was conducted using the second fraction of semen, which was collected from 15 male dogs, diluted to a total sperm count of 100x10(6) for each insemination with egg-yolk Tris (eyT) citrate acid buffer and incubated at 4 degrees C for 48 hours. Luteinizing hormone (LH) surge was detected to determine the optimal time for mating using canine LH assay kits. Artificial insemination using 100x10(6) sperm was performed on the fourth and sixth days or the fifth and seventh days after the LH surge. The conception rates were 33% (4/12) and 89% (8/9), respectively; the whelping rates also showed similar results. Serum LH and follicle stimulating hormone (FSH) concentrations were measured in nine dogs, and the mean LH concentration (+/- standard deviation) at LH surge was 15.77+/-7.66 ng/ml. The time of the LH surge detected by the canine LH assay kit was very similar to that measured by radioimmunoassay (RIA).     

Hysteroscopic insemination of mares with low numbers of nonsorted or flow sorted spermatozoa

The objectives of this study were 1) to compare pregnancy rates resulting from 2 methods of insemination using low sperm numbers and 2) to compare pregnancy rates resulting from hysteroscopic insemination of 5 x 106 nonsorted and 5 x 106 spermatozoa sorted for X- and Y-chromosome-bearing populations (flow sorted). Semen was collected with an artificial vagina from 2 stallions of known acceptable fertility. Oestrus was synchronised (June to July) in 40 mares, age 3-10 years, by administering 10 ml altrenogest orally for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. All mares were given 3000 iu hCG i.v. at the time of insemination to induce ovulation. Mares were assigned randomly to 1 of 3 treatment groups: mares in Treatment 1 (n = 10) were inseminated with 5 x 10(6) spermatozoa deposited deep into the uterine horn with the aid of ultrasonography. Mares in Treatment 2 (n = 10) were inseminated with 5 x 10(6) spermatozoa deposited onto the uterotubal junction papilla via hysteroscopic insemination. Mares in Treatment 3 (n = 20) were inseminated using the hysteroscopic technique with 5 x 10(6) flow sorted spermatozoa. Spermatozoa were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Pregnancy was determined ultrasonographically at 16 days postovulation. Hysteroscopic insemination resulted in more pregnancies (5/10 = 50%) than did the ultrasound-guided technique (0/10 = 0%; P<0.05) when nonsorted sperm were inseminated. Pregnancy rates were not significantly lower (P>0.05) when hysteroscopic insemination was used for sorted (5/20 = 25%) and nonsorted spermatozoa (5/10 = 50%). Therefore, hysteroscopic insemination of low numbers of flow sorted stallion spermatozoa resulted in reasonable pregnancy rates.     

Relationship between the sperm count and the fertilization rate of ova ovulated from the contralateral ovary in intrauterine horn insemination in cats

Unilateral intrauterine horn insemination (UIUI) was carried out in cats, and we investigated the fertilization rate of ova ovulated from the contralateral ovary. Various numbers of sperm were used to inseminate the uterine horn on the side where ovulation was inhibited. The rates of conception were 1/11 (9.1%), 2/11 (18.2%), and 5/7 (71.4%) in the 2 x 10(6), 4 x 10(6), and 8 x 10 (6) groups, respectively. Furthermore, the fertilization rate was 70.7% in the 8 x 10(6) group. Thus, ova ovulated from the contralateral ovary were not fertilized or the fertilization rate was low in some cats even when UIUI was performed with a large number of sperm.     

Feline embryo transfer during the non-breeding season

To obtain normal kits by embryo treansfer (ET) during the non-breeding season, maintenance of pregnancy was carried out by administration of sustained action progesterone (P4) in queens. Embryos were recovered six days after mating from five donor queens in which ovulation was induced by administration of eCG and hCG. The number of embryos recovered ranged from 24 to 53 (mean: 37.2 +/- 6.4) per animal and most embryos were compacted morulae. The yield of embryos was 49.0-93.3% (mean: 73.8 +/- 9.6%). As for recipients, porcine pituitary gland preparation and hCG were administered to 19 queens and estrus and ovulation were induced in 18 queens (94.7%). These queens underwent intrauterine ET of five compacted morulae and 17 cats (94.4%) were impregnated. The number of implantations was 2-5 (mean: 3.7 +/- 0.3). Among these impregnated queens, 15 cats received P4 adminstration starting on day 24 of gestation and 1-5 newborns (mean: 3.4 +/- 0.3) were obtained by normal delivery or caesarean section on day 64-69 of gestation. However, two animals that were not treated with P4 underwent spontaneous abortion about the mid gestational period. Therefore, it is possible to obtain normal kits from queens in the non-breeding season by ET with maintenance of pregnancy by P4 administration.     

Accessory sperm: their importance to fertility and embryo quality, and attempts to alter their numbers in artificially inseminated cattle.

Accessory sperm number and its relationship to fertilization and embryo quality was evaluated in cattle after nonsurgical recovery of ova or embryos 6 d after insemination. Efforts to alter accessory sperm number per ovum included 1) blockage of retrograde sperm loss at insemination using a modified insemination device, 2) elevated sperm number per inseminate (40 x 10(6) vs 20 x 10(6], and 3) alteration in semen quality (percentage of viable and morphologically normal sperm in the inseminate). None of these efforts affected accessory sperm number per ovum or embryo. However, blockage of retrograde semen flow for 3 h or use of semen of below-average quality (decreased percentage of viable and morphologically normal sperm) resulted in significant decreases in number of viable embryos and increases in number of degenerate embryos and unfertilized ova compared with conventional insemination (P less than .03) and use of semen with an average percentage of viable and morphologically normal sperm (P less than .06). Number of accessory sperm per embryo or ovum was positively related to fertilization and embryo quality (P less than .05). Mean accessory sperm +/- SD and the median value (in parentheses) for unfertilized ova, degenerate embryos, and embryos classified fair to poor and excellent to good were, respectively, .3 +/- .8 (0), 5.4 +/- 8.9 (1.0), 15.8 +/- 28.6 (3.5), and 16.9 +/- 29.5 (5.0). We conclude that efforts to improve accessory sperm numbers per embryo or ovum failed and that high variation and skewness of accessory sperm toward 0 may make median values more meaningful than means.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of boar exposure at time of insemination on factors influencing fertility in gilts

The effect of boar exposure during artificial insemination (AI) on semen backflow, fertilization, and embryo quality was evaluated. Gilts (approximately 170 d) were induced into estrus with PG600, and ovulation was synchronized using hCG 72 h later. Estrus detection was initiated after PG600 and continued at 12-h intervals. At estrus, gilts were allotted to receive boar exposure (BE, n = 20) or no boar exposure (NBE, n = 20) during AI. Gilts receiving NBE were identified to be in estrus prior to AI and the boar was then removed for 1 h, whereas gilts in the BE group received 15 min of exposure during AI. Insemination occurred in crates at 12 and 24 h after onset of estrus with 3 x 10(9) sperm/80 mL. Backflow was collected continuously with samples taken at time 0, (during AI), and at 0.25, 0.5, 0.75, 1, 2, 4, and 8 h after first and second AI. The effect of treatment was evaluated for time of insemination (min), backflow (mL), and sperm in backflow samples. Oviducts were flushed 2 d after first AI to evaluate the effect oftreatment on fertilization rate, accessory sperm numbers on embryos (scored 1 to 5), and embryo quality. There was no effect of first or second AI; therefore, data were pooled. Average duration of AI was 3.7 +/- 0.2 min and was not influenced by BE (P < 0.10). However, during the initial stage of AI, BE reduced the volume of semen (18.6 vs 32.4 +/- 3 mL) and the number of sperm lost (0.8 vs 1.3 +/- 0.15 x 10(9) sperm) compared to NBE (P < 0.05). There was a treatment x time effect (P < 0.05) for volume of backflow. By 45 min, the BE gilts lost more volume (9.0 vs 3.6 mL) compared to the NBE group, but sperm loss did not differ. Between 1 and 8 h after AI, neither volume nor sperm loss was influenced by treatment. By 8 h, total leakage (65 vs 63 mL) and total sperm loss (1.6 x 10(9) vs 1.8 x 10(9) sperm) were not influenced by BE (P > 0.10). However, more accessory sperm (P < 0.01) were found on embryos for the NBE (> or = 11 sperm/embryo) compared to BE embryos (< or = 10 sperm/embryo). Despite this observation, percentages of fertilized embryos (99.5 +/- 0.5 %) and number of embryos (11.5 +/- 0.1) were not different (P > 0.10). In conclusion, AI in the presence of a mature boar did not affect total semen leakage, sperm loss, fertilized embryos, or embryo quality. The importance of boar exposure during insemination was evident from less leakage during insemination, but had no effect on fertility; this suggests that the elimination of boar exposure during AI may not be deleterious to reproductive performance.     

Laparoscopic intra-uterine insemination of fallow deer with frozen-thawed or fresh semen after synchronisation with CIDR devices

This study investigated the efficacy of fixed-time laparoscopic intra-uterine insemination of farmed fallow deer (Dama dama) with frozen-thawed or fresh semen. In the trials with frozen-thawed semen, a total of 547 mature non-lactating does across five New Zealand farms were used. For oestrous synchronisation and artificial insemination, a standard control regimen was applied to at least 30% of the does on each farm, involving the insertion of single CIDR type-G devices intravaginally for 14 days, deposition of 50 x 10(6) frozen-thawed spermatozoa at 65 hours after withdrawal of the CIDR device and the continuous presence of vasectomised bucks from the insertion of the CIDR device until 10 days after insemination. Various aspects of this protocol were changed for the remaining does on each farm, including inseminations at 60 or 70 hours, the absence of vasectomised bucks, insemination with 25 x 10(6) or 10 x 10(6) spermatozoa, synchronisation with CIDR type-S devices and synchronisation with prostaglandin. The conception rate, based on rectal ultrasonography at 45 days after insemination, was 67% across all treatments (n=547). Corrected conception rates (+/-s.e.), calculated following between-farm adjustments, were 67+/- 3% for the control regimen, 67+/- 9% and 73 +/- 8% for inseminations at 60 and 70 hours respectively, 61 +/- 9% for absence of bucks, 80 +/- 8% and 74 +/- 9% for inseminations with 25 x 10(6) and 10 x 10(6) spermatozoa respectively, 62 +/- 10% for CIDR type-S device synchronisation, and 49 +/- 10% for prostaglandin synchronisation. Despite apparent differences, none of the treatments resulted in adjusted conception rates that were significantly different from the control regimen (P>0.01). In the trials with fresh semen, 216 does in the USA were inseminated at 69-71 hours after withdrawal of the CIDR device using either cryopreserved semen from New Zealand (n=158; 25 x 10(6) spermatozoa per inseminate) or fresh semen (n=58; 7.5 x10(6) to 20 x 10(6) spermatozoa per inseminate) collected less than 10 hours earlier. The overall conception rates were 77% and 81% respectively, with no significant differences between semen type (frozen v. fresh) or fresh spermatozoa number per inseminate (P>0.01). A further 102 does in New Zealand similarly received fresh semen from 3/4 Mesopotamian buck. Doses of 10 x 10(6) (n=35), 5 x 10(6) (n=32) or 2.5 x 10(6) (n=35) spermatozoa per inseminate were delivered at 69-71 hours after withdrawal of the CIDR device. The conception rates were 77%, 66% and 51% respectively, reflecting a dose effect (P<0.05). However, 1/4 Mesopotamian does in the group (n=19) exhibited higher conception rates (95% overall) irrespective of semen dose, possibly indicating a semen/recipient genotype interaction. It is concluded that laparoscopic intra-uterine insemination of fallow deer with frozen-thawed or fresh semen at fixed intervals after removal of a CIDR device can give acceptable conception rates under a range of on-farm management options and semen doses.     

Artificial insemination with frozen epididymal sperm in cats

Artificial insemination with frozen cauda epididymal sperm was performed in cats. Sperm were transmigrated from the epididymides in 10 male cats. The mean sperm motility and viability were 67% and 82.5%, respectively, and 11.6 x 10(7) sperm were recovered. The mean sperm motility after thawing was 24.0%. Eleven female cats received unilateral intrauterine insemination of 5 x 10(7) sperm, and the conception rate was 27.3% (3/11). This was the first case of conception obtained with frozen epididymal sperm in cats.     

Retrograde flow of spermatozoa into the urinary bladder of cats during electroejaculation, collection of semen with an artificial vagina, and mating

The effect of methoxamine on retrograde flow of spermatozoa into the urinary bladder of domestic cats during electroejaculation and the incidence of retrograde flow during the collection of semen with an artificial vagina, or during mating was examined. In experiment 1, urine collected by cystocentesis prior to electroejaculation was azoospermic or contained few, nonmotile spermatozoa, whereas urine collected after electroejaculation contained more (P = 0.002) spermatozoa, and motile spermatozoa were evident in urine obtained from 6 of 8 cats. Administration of methoxamine hydrochloride (200 micrograms/kg of body weight, IV) did not affect spermatozoal output or percentage of retrograde flow. Percentage of retrograde flow for control cats ranged from 61.18 to 92.95% (mean +/- SD, 80.00 +/- 14.28%) and for methoxamine-treated cats, ranged from 15.25 to 92.49% (mean +/- SD, 58.10 +/- 32.28%), but the difference was not significant. In experiment 2, an artificial vagina was used to collect semen from 5 of the 8 cats used in experiment 1. Urine collected by cystocentesis after ejaculation contained spermatozoa, and motile spermatozoa were evident in the urine from 4 of 5 cats. The mean (+/- SD) percentage of retrograde flow for these 5 cats was 46.82 +/- 31.67% (range, 14.56 to 90.32%). In experiment 3, each of the 5 cats that were used in experiments 1 and 2 were mated. Spermatozoa were recovered from the vagina of each mated female, and motile spermatozoa were also present in postejaculation urine obtained by cystocentesis from each of the 5 male cats. Mean total number of spermatozoa in the postmating urine was 29.42 +/- 33.58 x 10(6) (range, 0.22 x 10(6) to 76.05 x 10(6) spermatozoa).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Insemination–Ovulation Interval and Addition of Seminal Plasma on Sow Fertility to Insemination of Cryopreserved Sperm

In swine, the use of frozen-thawed (FT) sperm for artificial insemination (AI) is limited because of poor sow fertility, possibly associated with a post-thaw capacitation-like status resulting in fewer fully viable sperm. Sow fertility to AI with FT sperm may improve with deeper deposition of sperm within the female tract, insemination very close to ovulation, or reversal of cryocapacitation by seminal plasma (SP). We performed two experiments to examine these suggestions. In experiment 1, 122 multiparous Yorkshire sows received 600 IU equine chorionic gonadotrophin at weaning and 5 mg pLH 80 h later to control time of ovulation. The predicted time of ovulation (PTO) was 38 h after pLH injection. Thereafter, sows were assigned on the basis of parity to a single AI of FT sperm at 2 h before PTO, or at 12 h before PTO, or FT sperm supplemented with 10% SP at 12 h before PTO. Control sows received fresh semen at 12 h before PTO. All semen doses were adjusted to 3 x 10(9) live cells and deposited into the cervix. Experiment 2 employed 99 multiparous crossbred sows and repeated the treatments of experiment 1 except that all FT inseminations were intrauterine. In both experiments, farrowing rates were lower (p < 0.01) following FT inseminations with no effect of time of insemination or of supplemental SP. In experiment 1, litter size was smaller following FT insemination (p < 0.05), but no effect on litter size was evident in experiment 2. Supplemental SP had no effect on litter size in either experiment. The lack of effect of either SP or timing of FT insemination on sow fertility suggests that the non-lethal sperm cryoinjury affecting fertility involves more than just cryocapacitation.     

Influence of Sperm Cell Dose and Post-insemination Backflow on Reproductive Performance of Intrauterine Inseminated Sows

This study was carried out to evaluate the effects of the sperm cell dose and semen backflow on the pregnancy rate and number of embryos of sows inseminated once at 0-24 h before ovulation, using an intrauterine technique. The results were analysed from a total of 211 sows assigned to three groups inseminated with doses of 0.25 x 10(9) (T1), 0.5 x 10(9) (T2) and 1.0 x 10(9) (T3) spermatozoa. Semen backflow was observed in 95% of the females (143/151) evaluated for this purpose. The percentage of semen backflow is close to two-third of the volume and the percentage of sperm is around 15% of the infused sperm dose. Intrauterine insemination can be successfully performed provided that at least 0.5 billion of sperm cell dose is infused at an interval of 0-24 h before ovulation.     

Sperm migration in pigs after deep intrauterine and intraperitoneal insemination

Deep intrauterine insemination in pigs allows sperm deposition only into one uterine horn, but bilateral fertilization of oocytes occurs. How the sperm reach the contralateral oviduct remains disputable. The aim of this experiment was to study possible transperitoneal and/or transuterine sperm migration ways. Follicle growth and ovulation were induced in 24 peripubertal gilts with eCG and hCG 72 h after eCG. Endoscopic intrauterine insemination (IUI) was performed 32 h after hCG with 20 ml of extended semen (60 × 10(6) spermatozoa) as follows: Group CONTROL (n=8) received IUI into the right horn, and the left horn served as non-treated control; Group LIGATURE (n=8) received IUI into the right horn, and the left horn was closed by endoscopic double ligature close to the bifurcation; Group INTRAPERITONEAL (IPI; n=8) received IUI into the right uterine horn, the left horn was closed by double ligature and semen was deposited intraperitoneally at the surface of the left ovary. Genital tracts were removed 65-66 h after hCG, the oviducts were flushed and ova (n=299) were analyzed for fertilization and cleavage. Furthermore, the accessory spermatozoa count/oocyte was graded as 0, without spermatozoa, 1, <5 spermatozoa, 2, 5-50 spermatozoa, 3, 50-100 spermatozoa and 4, >100 spermatozoa. The results indicate that low dose IUI into one horn provides a lower grade of accessory spermatozoa in the contra-lateral side (1.6 vs. 2.8). No spermatozoa were found in ova flushed from oviducts of the ligated uterine horn, even after intraperitoneal insemination (P<0.05), and no fertilization occurred, respectively. Our results clearly indicate that after low dose IUI into one uterine horn, spermatozoa reach the contralateral oviduct via transuterine migration.     

Comparison between the Sperm Morphology in Semen Samples Obtained from Yearling Beef Bulls by Transrectal Massage of the Ampullae and Cauda Epididymal Dissection

As electroejaculation (EEJ) is prohibited for use on unanaesthetized animals in Sweden, there is a need for an alternative method of semen collection from bulls in the field. The aim of the present study was to evaluate the use of transrectal massage (TM) of the ampullae to collect semen from yearling beef bulls under field conditions in Sweden. Transrectal massage was performed on 52 yearling beef bulls. Volume of semen collected, duration of procedure, percentage progressively motile sperm, and sperm concentration were measured. Smears were prepared for sperm morphology examination. Semen samples were obtained from 47 of 52 bulls. Mean volume was 3.2 ml (SD +/- 3.7), mean duration of collection was 7.4 min (SD +/- 2.8), mean percentage progressively motile sperm was 43.5% (SD +/- 29.2) and mean concentration was 201.9 x 10(6) spermatozoa/ml (SD +/- 278 x 10(6)). Twenty-three of the 52 bulls were slaughtered 3-4 days after semen collection and aliquots of the cauda epididymal contents were collected for sperm morphology examination. The percentages of proximal droplets, abnormal tails and abnormal midpieces were significantly higher (p < 0.05) and the percentage of detached heads was significantly lower (p < 0.05) in the post-mortem samples compared with those in the TM samples. However, importantly there was no significant difference between the two sample types in the percentages of abnormal heads. This study demonstrates that semen can be collected from yearling beef bulls by TM. We think that TM constitutes a useful tool, when semen collection with EEJ or artificial vagina (AV) is not possible under field conditions, when included in the bull breeding soundness evaluation (BBSE) protocol. However, further studies are needed, and presently being carried out, to evaluate if semen samples collected by TM are comparable with semen samples collected by AV.     

Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen-thawed spermatozoa onto the uterotubal junction

REASONS FOR PERFORMING STUDY: To compensate for the wide variation in the freezability of stallion spermatozoa, it has become common veterinary practice to carry out repeated ultrasonography of the ovaries of oestrous mares in order to be able to inseminate them within 6-12 h of ovulation with a minimum of 300-500 x 10(6) frozen-thawed spermatozoa. Furthermore, in order to achieve satisfactory fertility, this requirement for relatively high numbers of spermatozoa currently limits our ability to exploit recently available artificial breeding technologies, such as sex-sorted semen, for which only 5-20 x 10(6) spermatozoa are available for insemination. OBJECTIVES: This study was designed to evaluate and compare the efficacy of hysteroscopic vs. conventional insemination when low numbers of spermatozoa are used at a single fixed time after administration of an ovulation-inducing agent. METHODS: In the present study, pregnancy rates were compared in 86 mares inseminated once only with low numbers of frozen-thawed spermatozoa (3-14 x 10(6)) at 32 h after treatment with human chorionic gonadotrophin (hCG), either conventionally into the body of the uterus or hysteroscopically by depositing a small volume of the inseminate directly onto the uterotubal papilla ipsilateral to the ovary containing the pre-ovulatory follicle. RESULTS: Pregnancy rates were similarly high in mares inseminated conventionally or hysteroscopically with 14 x 10(6) motile frozen-thawed spermatozoa (67% vs. 64%). However, when the insemination dose was reduced to 3 x 10(6) spermatozoa, the pregnancy rate was significantly higher in the mares inseminated hysteroscopically onto the uterotubal junction compared to those inseminated into the uterine body (47 vs. 15%, P < 0.05). CONCLUSIONS: When inseminating mares with <10 x 10(6) frozen-thawed stallion spermatozoa, hysteroscopic uterotubal junction deposition of the inseminate is the preferred method. POTENTIAL CLINICAL RELEVANCE: Satisfactory pregnancy rates are achievable after insemination of mares with frozen-thawed semen from fertile stallions 32 h after administration of human chorionic gonadotrophin (Chorulon). Furthermore, these results were obtained when mares were inseminated with 14 x 10(6) progressively motile frozen-thawed spermatozoa from 2 stallions of proven fertility.     

Distribution and retention of spermatozoa with acrosomal and nuclear abnormalities in the cow genital tract

Two experiments were conducted to evaluate the fate of sperm following uterine insemination. In Exp. I, five pairs of Holstein cows were inseminated with egg yolk-Tris extended semen (approximately 1.0 X 10(9) sperm; .5 ml) from five ejaculates from a single bull that had high levels (approximately 70%) of morphologically abnormal sperm. Cows were slaughtered 12 h after insemination. The genital tracts were removed and promptly clamped into defined regions. Sperm were recovered by flushing with 2.9% sodium citrate buffer. Proportions of abnormal sperm in the various regions were compared with those in the inseminate. Sperm numbers were also determined from each region. Regions of the tract varied in number of sperm (P less than .001), proportions of knobbed acrosomes (P less than .001), tapered heads (P less than .001), protoplasmic droplets (P less than .001), tail abnormalities (P less than .029) and total abnormalities (P less than .002). A total of 63.5 +/- 6.4 X 10(6) sperm was recovered. These sperm were distributed throughout the tract as follows: vagina, 91.8%; cervix, 5.4%; uterine horns, 2.7%, and uterotubal junctions-isthmi, .04%. No sperm were recovered from ampullae. Because retrograde movement of sperm from the uterus occurred in Exp. I, we conducted Exp. II to determine the extent of sperm loss from the genital tract following insemination. Three pairs of Holstein cows were inseminated with .42 X 10(9) sperm (.5 ml; egg yolk-Tris extender) from the same bull used in Exp. I (three ejaculates). All discharged mucus and urine was collected for 12 h after insemination for recovery of sperm. Aspirates (approximately 1 ml) of mucus from the vagina were evaluated during the 12-h post-insemination period for numbers of sperm and leucocytes. Sperm were also recovered from the tract following slaughter (approximately 12 h) to determine retention. Overall, 73 +/- 3.7% of inseminated sperm were recovered. Components were: inseminate lost from the genital tract in discharged mucus, 60 +/- 4.6%; lost in urine, .06 +/- .02%; aspirated from the vagina, 4.4 +/- 1%; adhered to equipment, 1.3 +/- .3%, and retained in the genital tract, 6.5 +/- 1.6%. Predicted numbers of sperm contained in discharged mucus 2 h post-insemination were greater (P less than .009) than at subsequent hours.(ABSTRACT TRUNCATED AT 400 WORDS)     

猫人工授精技术研究进展

人工授精技术是一种较先进的辅助生殖技术,可提高繁殖育种的效益,但因猫类较独特的生殖特点,其人工授精技术发展受限。近年来,科研人员和临床工作者借鉴其他物种相对成熟的人工授精技术,结合猫类生殖特点,对猫人工授精过程中精液采集、精液品质评估、精液保存、刺激母猫排卵方式及具体的人工输精等相关技术进行了优化和创新,如开发了新颖实用的尿导管法采精技术以采集猫精液;在传统精液品质评估的基础上,配合使用荧光染色技术、流式细胞技术及体外受精技术对猫精液品质进行评估;探索不同冷冻液配方及冷冻程序对猫精液保存效果的影响,并尝试使用超快速冷冻法保存公猫遗传物质;研究使用不同种类及不同剂量的激素对诱导母猫发情和排卵效果的差异,以及不同输精部位、方法及时机对母猫妊娠结果的影响等。尽管其中一些新技术与方法已在试验动物中彰显出了其实际应用潜力,但当下依旧还存在操作相对复杂、成本较高、效果低于预期等问题,导致目前猫人工授精技术的应用较局限,不如在犬或其他动物中应用广泛,未来还需更进一步研究和探索才能满足临床实际应用的需要。文章就上述猫人工授精技术中各个方面的研究进展进行了综述,以期为该技术的进一步研究和应用提供参考。     

Effect of Sperm Cryopreservation and Supplementing Semen Doses with Seminal Plasma on the Establishment of a Sperm Reservoir in Gilts

Frozen-thawed (FT) boar sperm have a reduced fertile life, due in part to a capacitation-like status induced by cooling. Reversal of this cryocapacitation in vitro by exposure to boar seminal plasma (SP) has been demonstrated. The objective of these studies was to determine the effect of SP on the ability of FT sperm to create an oviductal sperm reservoir following artificial insemination (AI). In Experiment one, 35 pre-pubertal gilts were injected (IM) with 400 IU eCG plus 200 IU hCG to induce oestrus. At detection of oestrus, gilts were inseminated with 3 x 10(9) live sperm, either fresh (FS; n = 13), FT (n = 10), or FT supplemented with 10% v/v SP (n = 12). Gilts were killed 8 h later, their reproductive tracts recovered and the uterotubal junctions (UTJs) flushed to recover sperm. Fewer (p < 0.01) sperm were recovered following FT, compared to FS, inseminations, and there was no evident effect of SP. In Experiment two, 30 pre-pubertal gilts received IM injections of 1000 IU eCG followed by 5 mg pLH 80 h later to control time of ovulation. Gilts were inseminated with 3 x 10(9) live FS sperm (n = 6), FT sperm (n = 15) or FT sperm plus 10% SP (n = 9) at 12 h before ovulation and then sacrificed 8 h later. The UTJs were dissected and flushed for sperm recovery. Fewest (p < 0.001) sperm were recovered following FT insemination and there was no evident effect of SP. These data demonstrate that the size of the sperm reservoir is markedly reduced in gilts inseminated with FT sperm. However, the lack of effect of SP suggested that either it did not reverse cryocapacitation or that such a reversal does not impact the in vivo ability to create a sperm reservoir.