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.     

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.     

Effect of the previously gravid uterine horn and postpartum interval on follicular diameter and conception rate in beef cows treated with estradiol benzoate and progesterone

An experiment was performed to evaluate the effect of the side of ovulation with respect to the previously gravid uterine horn on fertility of cows inseminated at one of two periods postpartum. All cows were treated with an intravaginal progesterone insert for 7 d and received estradiol benzoate (2 mg, i.m.) at the time of device insertion, prostaglandin F2alpha (25 mg, i.m.) at the time of device removal, and estradiol benzoate (1 mg, i.m.) 30 h after device removal. All cows were inseminated 28 to 30 h after the second treatment with estradiol benzoate, regardless of observed estrus. Cows treated in Period 1 received inserts at 16 to 20 d postpartum and were inseminated at 25 to 29 d postpartum. Cows treated in Period 2 received inserts at 26 to 30 d postpartum and were inseminated at 35 to 39 d postpartum. Diameter of the largest follicle at insert removal was greater in cows treated in Period 2 (10.1 +/- 0.3; mm +/- SEM) than in cows treated in Period 1 (9.1 +/- 0.3; P < .05). Diameter did not differ with the side of ovulation in respect to the previously gravid uterine horn. Diameter was greater in cows 5 to 9 (10.3 +/- 0.3) than in cows 3 to 4 (9.0 +/- 0.3) or 10 to 13 (9.4 +/- 0.6) yr of age (P < .01). The proportion of cows that ovulated from the ovary contralateral to the previously gravid uterine horn was greater (P < .05) than of those that ovulated from the ipsilateral ovary, and the incidence of ovulation was reduced in cows 3 to 4 yr of age (P < .01). Conception rate tended to be greater for ovulation from the ipsilateral compared with the contralateral ovary, relative to the previously gravid uterine horn (P < .10) and for ovulation from the right than the left ovary (P < .06). Conception rate was less if cows ovulated a follicle that was < 9 mm than a follicle > or = 9 mm in diameter at insert removal (P < .01) and was greater in cows inseminated in June than in April or May (P < .05). In conclusion, in cows in which estrus was synchronized at 25 to 39 d postpartum, ovulation from either the ovary ipsilateral to the previously gravid uterine horn, or the right ovary, tended to increase fertility.     

Induction of estrus and ovulation in the bitch, using exogenous gonadotropins

The capability of pregnant mare serum (PMS) and human chorionic gonadotropin (HCG) to induce estrus and ovulation was tested in mature, anestrous bitches. The PMS was given for 10 consecutive days in 1 of 3 regimens: 500 IU/day (experiment 1), 250 IU/day (experiment 2), or 20 IU/kg/day (experiment 3). The HCG was given as a single 500-IU dose on experimental day 10. Controls were given saline solution. Vaginal smears were collected on days 1, 3, 5, 7, 9, and 12 by jugular venipuncture, and the plasma was assayed for progesterone concentration by radioimmunoassay. On day 13, the bitches were euthanatized, ova were flushed from the uterine tubes (oviducts), and the ovaries were collected and prepared for microscopic examination. Fourteen of 25 bitches treated with PMS and HCG showed estrus and ovulated. Proestrus (vaginal bleeding) commenced between experimental days 7 and 10. Estrus commenced on day 9 or 10. Progesterone increased from approximately 1 ng/ml on day 1 to more than 6 ng/ml on day 12. Numbers of ovulation sites on both ovaries were 4.7 +/- 1.1 and 4.6 +/- 0.5 (mean +/- SEM) in those given the daily doses of 500 and 250 IU of PMS and 9.8 +/- 1.5 in experiment 3 bitches. Eleven hormone-treated dogs and 7 saline-treated dogs did not show any detectable response. Neither cystic nor unovulated, luteinized follicles appeared on the ovaries.     

Distribution of Spermatozoa and Embryos in the Female Reproductive Tract after Unilateral Deep Intra Uterine Insemination in the Pig

The present study was performed to investigate the number of either the spermatozoa or the embryos in the reproductive tracts of sows after unilateral, deep, intra uterine insemination (DIUI). Two experiments were conducted, 10 sows were used in experiment I and eight sows were used in experiment II. Transrectal ultrasonography was used to examine the time when ovulation took place in relation to oestrus behaviour. The sows were inseminated with a single dose of diluted fresh semen 6-8 h prior to expected ovulation, during the second oestrus after weaning. In experimental I, five sows were inseminated by a conventional artificial insemination (AI) technique using 100 ml of diluted fresh semen, containing 3000 x 10(6) motile spermatozoa and five sows were inseminated by the DIUI technique with 5 ml of diluted fresh semen, containing 150 x 10(6) motile spermatozoa. The sows were anesthetized and ovario-hysterectomized approximately 24 h after insemination. The oviducts and the uterine horns on each side of the reproductive tracts were divided into seven segments, namely ampulla, cranial isthmus, caudal isthmus, utero-tubal junction (UTJ), cranial uterine horn, middle uterine horn and caudal uterine horn. Each segment of the reproductive tracts was flushed with Beltsville thawing solution (BTS) through the lumen. The total number of spermatozoa in the flushing from each segment were determined. In experimental II, eight sows were inseminated by the DIUI technique using 5.0 ml diluted fresh semen containing 150 x 10(6) motile spermatozoa. The sows were anesthetized 61.1 +/- 12 h after insemination (48-72 h) and the embryos were flushed from the oviduct through the proximal part of the uterine horn. It was revealed that, in experimental I, the spermatozoa were recovered from both sides of the reproductive tract in the AI-group, and from unilateral side of the reproductive tract in the DIUI-group (three sows from the left and two sows from the right sides). The number of spermatozoa recovered from the reproductive tracts was higher in the AI- than the DIUI-group (p < 0.001). In experiment II, fertilization occurred in five of eight sows (62.5%) after DIUI. The number of ova that ovulated were 16.4 +/- 2.6 per sow and the embryos numbering 11.4 +/- 2.3 per sow were recovered from both sides of the reproductive tract. In conclusion, the spermatozoa given by DIUI could be recovered from only one side of the reproductive tract of sows at approximately 24 h after DIUI via the flushing technique. However, embryos were found in both sides of the oviducts and the proximal part of the uterine horns 48-72 h after insemination, indicating that the fertilization occurred in both sides of the oviducts.     

Factors affecting transuterine migration of canine embryos

Dogs, cats, and pigs have a bicornuate uterus, and transuterine migration of embryos occurs in 40% or more of pregnant animals. However, the mechanism of the transuterine migration has not been elucidated in dogs. Thus, we investigated the occurrence of transuterine migration of embryos when embryos were retained in an unilateral uterine tube with more ovulated ova (Experiment 1), when one ovary was excised (Experiment 2), and when ova ovulated from the right and left ovaries were fertilized with sperm from male dogs with different blood types (Experiment 3). Transuterine migration of embryos was observed in 7/8 (87.5%), 10/10 (100%), and 11/17 (67.4%) fertilized animals in Experiments 1, 2, and 3, respectively. In Experiment 3, intrauterine embryo mixing reported in pigs did not occur. These findings suggest that transuterine migration of embryos occurs due to the number of embryos that enter the uterus but that differences in the number of ovulated ova between the right and left ovaries or the number of embryos retained in the uterine tube do not affect the migration.     

Effect of unilateral cornual insemination upon fertilization rate in superovulating and single-ovulating cattle

In Exp. 1, 21 first-service cattle and seven repeat-breeder cattle, averaging 4.7 infertile services, were brought into estrus and superovulated by treatment with follicle-stimulating hormone and prostaglandin F2 alpha. At insemination, semen was deposited in the greater curvature of one uterine horn, about midway between the utero-cervical junction and the utero-tubal junction. Cattle were necropsied 2 to 7 d after estrus and ova were recovered and examined. The fertilization rate for first-service cows was 74% of 362 intact ova and for repeat-breeders, 43% of 128 intact ova (P less than .001). Fertilization rate in first-service cows was 81% on the side of semen deposition and 68% on the opposite side (P less than .01); the rates in repeat-breeders were 54% and 32% (P less than .025). Differences between sides were due mostly to four cows that averaged 93% fertilization on the side of semen deposition and 19% on the opposite side. The proportion of fertilized ova with accessory sperm (17%) did not differ between sides of the reproductive tract. In Exp. 2, 60 first-service and 32 repeat-breeder cows in natural estrus had semen deposited in the uterine body or in the greater curvature of one uterine horn, either on the side of impending ovulation or on the opposite side. At necropsy, 55 ova were recovered from first-service cows, of which 42 (76%) were intact and 13 (24%) were ruptured or fragmented. Of the 42 intact ova, 41 (98%) were cleaved. From the 32 repeat-breeders, 30 ova were recovered, of which 26 (87%) were intact and 4 (13%) were ruptured; 23 of the 26 intact ova (88%) were cleaved. Site of semen deposition had no significant effect on either fertilization rate or number of accessory sperm in either type of cow. First-service cows averaged more accessory sperm (40) than did repeat-breeders (19, P less than .01). Overall results indicated that sperm deposited deep in one uterine horn fertilized ova nearly as frequently in the opposite oviduct as in the adjacent oviduct except in 14% of superovulating cattle.     

Transport of spermatozoa in the reproductive tract of the bitch: observations through uterine fistula

Using unilateral uterine fistulas, the time required for spermatozoa to reach the end of the fistula after natural mating, artificial insemination (AI) in a normal standing posture (NP), and AI standing on the head (SH) was investigated in each of three stages of estrus. Conceptivity in these bitches was also investigated. Five experimental bitches were tested during a total of 8 estrous periods. The results are as follows; the time required for spermatozoa to reach the end of the fistula was almost the same in the early and middle stages, i.e., 30 sec to 1 min after natural mating and SH and less than 2 min for half the bitches in NP, although no intrauterine transport could be observed in the other half. In most cases of mating during the late stage no spermatozoa were found after any of the 3 methods of insemination. Five animals became pregnant in these experiments, but the other three failed to conceive. The implantation of fertilized ova occurred also in the fistulated uterine horn in all cases of pregnancy.     

Nature of early reproductive failure caused by bovine viral diarrhea virus

A 2-part study was undertaken to determine the effect of bovine viral diarrhea (BVD) virus on fertilization and early development of embryos. In experiment 1, 10 seronegative cows were superovulated and artificially inseminated twice during estrus. After the second insemination, 5 of the cows received intrauterine infusion of BVD virus suspension. The other 5 cows received suspending medium only and served as controls. All 10 cows were slaughtered on day 3, and ova and embryos were collected for morphologic evaluation. A total of 49 and 52 ova and embryos were collected from the control and virus-treated cows, respectively. Among the ova and embryos collected from control cows, 81.6% were fertilized, whereas only 52% were fertilized in the virus-treated group. The statistically significant difference (P less than 0.01) indicated that the virus interferes with fertilization. In experiment 2, the protocol was identical except for slaughter on day 13. Seventy-nine ova and embryos were collected from the 6 control cows, and the 6 virus-treated cows yielded 59 ova and embryos. Of the total ova and embryos recovered on day 13, 88.6% and 50.8% were hatched and developing normally in the control and virus-treated groups, respectively. The difference was highly significant (P less than 0.001). Unfertilized ova and degenerating embryos could not be differentiated on the basis of morphologic appearance. The nearly identical percentages of unfertilized ova in experiment 1 and unhatched ova and embryos in experiment 2 strongly suggested that fertilization failure is the principal manifestation of the observed adverse effect of BVD virus infection.     

Use of pulsatile intravenous administration of gonadotropin-releasing hormone to induce fertile estrus in bitches

The pulsatile IV administration of gonadotropin-releasing hormone (GnRH) was evaluated as a method to induce fertile estrus in 8 anestrous Beagle bitches. Bitches received 1.25 micrograms of GnRH every 90 minutes for 11 to 13 days. Gonadotropin-releasing hormone was delivered by use of an automatic pump. Reproductive history was known for all bitches, 4 of which, on the basis of 3 or 4 preceding cycles, had an interestrous interval of 219 +/- 14 days (mean +/- SEM). Estrus induction was attempted during early anestrus in 6 bitches (ie, 148 +/- 10 days since the preceding estrus) and late anestrus in 1 bitch (ie, 260 days since the preceding estrus); another bitch had not had an estrous cycle for nearly 2 years before GnRH administration. Signs of estrus were seen within 16 days after the start of GnRH administration in the bitches with regular estrous cycles (group 1, n = 7), and within 23 days in the bitch (group 2) with prolonged anestrus. All bitches were bred, and 7 of 8 (87.5%) became pregnant, with a mean litter size of 4.5 +/- 0.75. A normal hormonal response pattern was observed in group-1 bitches--a peak increase in plasma estrogen concentration of 22.3 +/- 2 pg/ml immediately before the onset of estrus. Peak plasma progesterone concentration (17.3 +/- 3 ng/ml) was observed 1 to 14 days after the onset of diestrus in the group-1 bitches that ovulated, and adequate plasma progesterone concentration was maintained throughout gestation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Influencing Factor of In Vitro Fertilization and Embryonic Transfer in the Domestic Fowl (Gallus domesticus)

This study was conducted to explore the influencing factors of ova in vitro fertilization (IVF) and transfer of the fertilized ova into the oviduct of recipient hens. The efficiency of fertilization was compared using three aspects: (i) the different time of ova collection and transfer, (ii) egg‐laying period of recipient hen; and (iii) semen volume. The following results are observed: 72%, 40% and 0% of ova were found in ovarian sac in 30～40 min, 50～60 min and more than 90 min post‐oviposition, respectively; 20%, 18%, 14% and 5.8% of ova were fertilized with 0.1, 0.2, 0.5 and 1.0 ml semen, respectively; and 33% and 100% of healthy chickens were hatched from fertile ova with 0.1 and 0.5 ml of semen, respectively. All oocytes obtained from ovary and mid‐oviduct were unfertilized. Embryos were transferred into recipient hens 30 min ± 10 min post‐oviposition, and 70% of shelled eggs were produced. There were no eggs produced in the other transfer times. This demonstrated that live chicken can be obtained by IVF of ova collected shortly after oviposition. It was important that the ovum was transferred into the oviduct infundibulum of recipient hens immediately or shortly after oviposition.     

Induction of oestrus by administering Inhibin antiserum along with equine chorionic gonadotropin in anoestrous bitches

As dogs experience oestrus only once or twice a year, it is necessary to establish an effective method of oestrous induction for efficient breeding. In the present study, we evaluated inhibin antiserum (IAS) on oestrous induction in anoestrous females. Bitches were administered 0.5 ml/kg IAS or a mixture of 50 IU/kg equine chorionic gonadotropin (eCG) and 0.5 ml/kg IAS and 500 IU human chorionic gonadotropin (hCG) administered 7 days after the mixture injection. As a control, bitches received 50 IU/kg eCG, with 500 IU hCG administered 7 days after eCG injection. Blood-tinged vaginal discharge, vulvar swelling, plasma progesterone concentrations and ovarian follicular development were assessed from day 0 to day 14. IAS alone injection did not induce oestrus in bitches at the anoestrous stage. Conversely, vulvar swelling, blood-tinged vaginal discharge and an estimated luteinizing hormone (LH) surge appeared on days 3–7, days 3–6 and days 7–9 after the IAS+eCG mixture injection, respectively, in all five bitches at the anoestrous stage. The average number of developing and ovulated follicles in bitches administered IAS+eCG was 8.8 and 9.6 respectively. A single eCG injection followed by hCG induced oestrous signs, with an average of 8.3 developing follicles and 4.5 ovulated follicles. This study revealed that IAS alone did not induce oestrus, but when IAS was used in combination with eCG, it induced oestrus and promoted a considerable number of ovulations in anoestrous dogs.     

Influence of the semen deposition site on the calves' sex ratio in Simmental dairy cattle

It has been suggested that the time of insemination has effect on the calves' sex ratio because of the differences in timing of capacitation, motility and survival time of the X and Y spermatozoa in the female reproductive tract. We have conducted a field trial to study the effects of different semen deposition sites on the sex ratio and fertility in cattle. Two groups of 450 cows were inseminated via artificial insemination: group A was inseminated into the uterine body and group B was inseminated deep into the uterine horn ipsilateral to the ovary with dominant follicle. After applying several exclusion criteria, a total of 607 pregnant cows were considered for data analysis (group A = 318 and group B = 289 cows). The conception rate was 7% higher (p < 0.05) in the group A, with 23% more of the male calf pregnancies (p < 0.001). At the same time, 18% more of the female calves were calved in the group B (p < 0.005). The difference in male calves between the two groups was 21% and in female calves was 20% (p < 0.001). We conclude that semen deposition site plays a significant role in differences in gender ratio observed after calving. Intracornual semen deposition resulted in a higher ratio of female calves, whereas uterine body deposition site resulted in higher male calves ratio, probably contributing physiologically to the differences in motility, capacitation time, the lifespan of X vs Y spermatozoa and to the pronounced shift of X spermatozoa in the female genital tract.     

Effect of dietary crude-protein type on fertilization and embryo quality in dairy cattle

An experiment was conducted to determine whether balancing dietary crude protein for optimal rumen degradability would improve fertilization rate and quality of ova in lactating dairy cows. Thirty-eight Holstein cows in early lactation were fed 1 of 2 diets formulated to be isocaloric and isonitrogenous, containing 16% crude protein. Diet 1 contained 73% rumen degradable intake protein, whereas diet 2 contained 64% rumen degradable intake protein. The cows were induced to superovulate and were inseminated, and ova were recovered nonsurgically on postbreeding day 7. Ova were counted and classified as fertilized or unfertilized. Fertilized ova were scored as excellent, good, fair, poor, or degenerate. Unfertilized ova and poor and degenerate embryos were considered to be nontransferable ova and excellent, good, and fair embryos were considered to be transferable ova. There were no differences for mean number of fertilized, unfertilized, transferable, or nontransferable ova recovered from cows fed the 2 diets (P greater than 0.10). Mean percentage of fertilized ova recovered from cows was greater (P less than 0.05) in those fed diet 2, compared with diet 1. Mean percentage of transferable ova recovered from cows tended to be greater (P = 0.06) in those fed diet 2, compared with diet 1. More cows failed to yield transferable ova (P less than 0.05) when fed diet 1, compared with diet 2. Fertilization failure or early degeneration of embryos may occur in cows fed excess rumen degradable protein.     

Effect of trypsin in semen on in vivo fertilization and early embryonic development in superovulated heifers

The effect of trypsin on the fertilizing capacity of bull semen was investigated as part of the evaluation of the addition of trypsin to semen as a method for destroying or inactivating infectious agents. Parts of the ejaculates from four bulls were treated with 0.3% trypsin solution. Both the treated and untreated aliquots of semen were frozen, thawed and used for the artificial insemination of superovulated heifers. Two hundred and thirty ova and embryos were collected from 22 heifers on day 7 after oestrus (insemination). One hundred and ten out of 164 (67%) embryos and ova from 15 heifers inseminated with trypsin-treated semen were classified as of transferable quality compared to 46 out of 66 (70%) in the control group of 7 heifers (p>0.05). There was no difference in the proportion of fertilized ova or degenerated embryos resulting from the control or trypsin-treated samples of frozen-thawed semen, which is consistent with results obtained previously using fresh semen.     

Distribution and viability of spermatozoa in the canine female genital tract during post-ovulatory oocyte maturation

Background

Unlike other domestic mammals, in which metaphase-II oocytes are ovulated, canine ovulation is characterized by the release of primary oocytes, which may take 12 to up to 36 hours. Further 60 hours are needed for maturation to secondary oocytes which then remain fertile for about 48 hours. Oestrus takes 7 to 10 days on average and may start as early as a week before ovulation. This together with the prolonged process of post-ovulatory oocyte maturation requires an according longevity of spermatozoa in the female genital tract in order to provide a population of fertile sperm when oocytes have matured to fertilizability. Therefore the distribution and viability of spermatozoa in the bitch genital tract was examined during post-ovulatory oocyte maturation.

Methods

Thirteen beagle bitches were inseminated on the day of sonographically verified ovulation with pooled semen of two beagle dogs containing one billion progressively motile spermatozoa. Ovariohysterectomy was performed two days later (group 1, n = 6) and four days later (group 2, n = 7). The oviduct and uterine horn of one side were flushed separately and the flushing’s were checked for the presence of gametes. The oviducts including the utero-tubal junction and the uterine horns, both the flushed and unflushed, were histologically examined for sperm distribution.

Results

The total number of spermatozoa recovered by flushing was low and evaluation of viability was limited. Prophase-I oocytes were collected from oviduct flushing in group 1, whereas unfertilized metaphase-II oocytes were detected in group 2. From day 2 to day 4 after ovulation a significant decrease in the percentage of glands containing sperm (P<0.05) and a marked reduction of the mean sperm number in uterine horn glands were observed. A concomitant diminution of spermatozoa was indicated in the utero-tubal junction accompanied by a slight increase in sperm numbers in the mid oviduct.

Conclusions

Oocyte maturation to metaphase-II stage is accompanied by a continuous sperm detachment and elimination in the uterine horns. Entrance of spermatozoa into the caudal oviduct seems to be steadily controlled by the utero-tubal junction thus providing a selected sperm population to be shifted towards the site of fertilization when oocyte maturation is completed.     

Conception rates in early postpartum ewes bred naturally or by intrauterine insemination

Ewes were treated with a medroxyprogesterone acetate (MAP) sponge for 8 d followed, at sponge removal, with 500 IU pregnant mare serum gonadotropin (PMSG) at d 30, 40 or 50 (d 0 = lambing) to induce estrus. Dry and lactating ewes were divided into equal numbers at each postpartum day and bred at estrus. Conception rates and number of accessory sperm were determined by flushing the oviducts 3 d after mating and examining the recovered ova. There was no effect (P greater than .05) of lactational status on conception rates. Conception rates increased (P less than .05) from d 30 (10%) to d 40 (45%) and from d 40 to d 50 (80%). There were fewer (P less than .05) ova with accessory sperm (5/26) in d-30 ewes compared with d-40 (10/27) or d-50 (12/24) ewes. In Exp. 2, ewes were assigned to two groups after receiving PMSG on d 30: 1) mated naturally or 2) inseminated during laparotomy near the uterotubal junction (UTJ). Dry and lactating ewes were divided evenly within each of the two treatments. Oviducts were flushed and ova were examined for cleavage. The conception rate was 60% in ewes that were inseminated in the UTJ vs 10% in ewes mated to rams (P less than .05). Lactational status had no effect on results. In conclusion, conception rates in postpartum ewes treated with MAP sponge and PMSG increased from postpartum d 30 to d 50 with natural breeding, and d-30 conception rates were increased over natural mating by insemination into the uterine horn near the UTJ.     

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.     

Method for obtaining ovine uterine secretions from unilaterally pregnant ewes

Development of the ovine conceptus was confined to the uterine horn ipsilateral to the corpus luteum (CL) by placing a ligature around that uterine horn at a point near the uterine body on day 5 of pregnancy. On day 140 of gestation, seven of 10 ewes were still pregnant and from 21 to 815 ml of uterine fluid (488 +/- 94 ml, X +/- SEM) were collected from the nongravid uterine horn. Total recoverable protein (X +/- SEM) was 13.4 +/- 3.4 grams. Polyacrylamide gel electrophoresis of the reduced proteins in presence of sodium dodecyl sulfate indicated that protein composition of uterine fluid was distinct from that of colostrum, serum, amniotic fluid, and allantoic fluid, and revealed the presence of two major polypeptides with molecular weights of about 57,000 and 58,500, respectively, plus numerous other minor components. Gel filtration on columns of Sephadex G-200 and Sepharose CL-6B suggested that these polypeptides formed a series of aggregates of high molecular weight when kept under nonreducing conditions. Glucose (.18 +/- .03 mg/ml), but not fructose, was present in uterine fluid. In addition, high levels of prostaglandin F (451.4 +/- 83.3 ng/ml) were present.     

Fertility after different artificial insemination methods using a synthetic semen extender in sheep

The present study aimed to investigate the fertility of ewes artificially inseminated with three different methods using a synthetic semen extender, AndroMed. The three methods of artificial insemination (AI) were cervical AI with fresh-diluted or frozen-diluted semen at observed estrus, and an intrauterine AI with frozen-thawed semen. A total of 80 ewes were treated with a controlled internal drug release (CIDR) containing 0.3 g progesterone per device for 12 days. In Experiment 1 (26 Suffolk ewes), superovulation was induced with 20 mg follicle-stimulating hormone and 250 IU equine chorionic gonadotropin (eCG) two days and one day before CIDR removal, respectively, during the non-breeding season. In Experiment 2 (54 Suffolk and Suffolk crossbred ewes), an intramuscular injection of 500 IU eCG was administered one day before CIDR removal to synchronize estrus and ovulation during the breeding season. In Experiment 1, fresh-diluted or frozen-thawed semen was deposited into the cervical orifice after estrus detection, and an intrauterine AI with frozen-thawed semen was performed by laparoscopy at a fixed-time basis without estrus detection. Embryos were recovered by uterine flushing 6 days after AI, and the rates of recovered, fertilized (cleaved) ova and embryos at the morula or blastocyst stage were compared among the three AI methods. In Experiment 2, the pregnancy rates after the three AI methods were compared. In Experiment 1, the rates of recovered ova were not significantly different among the three AI methods (52.5-56.7%). The rate of fertilized ova (81.0%) by laparoscopic AI with frozen-thawed semen was significantly higher compared with cervical AI of fresh-diluted (25.5%) or frozen-thawed (3.5%) semen, but the rate of embryos at the morula or blastocyst stage (17.6%) was significantly lower than that of the cervical AI with fresh-diluted semen (69.2%). The rates of ewes yielding fertilized ova were not significantly different among the three groups (44.4, 11.1 and 62.5% for cervical AI with fresh-diluted and frozen-thawed semen and intrauterine AI with frozen-thawed semen). In Experiment 2, the pregnancy rate of ewes intrauterinally inseminated with frozen-thawed semen (72.2%) was significantly higher than those of ewes inseminated cervically with fresh-diluted (5.5%) or frozen-thawed (0.0%) semen. The present results showed that acceptable fertilization and pregnancy rates could be obtained by an intrauterine AI with frozen-thawed semen using a synthetic semen extender (AndroMed), but not sufficient by the cervical AI with either fresh or frozen semen.