Oocyte transfer in mares with intrauterine or intraoviductal insemination using fresh, cooled, and frozen stallion semen

The objectives were to compare embryo development rates after oocyte transfer with: (1) intrauterine or intraoviductal inseminations of fresh semen versus intraoviductal insemination of frozen semen; (2) intraoviductal versus intrauterine inseminations of cooled semen. In Experiment I, oocytes were transferred into the oviduct, and recipients were inseminated into the uterus with 1 x 10(9) fresh spermatozoa, or into the oviduct with 2 x 10(5) fresh or frozen-thawed spermatozoa. In Experiment II, semen was cooled to 5 degrees C before intrauterine insemination with 2 x 10(9) spermatozoa or intraoviductal inseminations of 2 x 10(5) spermatozoa (deposited with the oocytes). In Experiment I, embryo development rates were similar (P>0.05) for intrauterine versus intraoviductal inseminations when fresh semen was used (8/14, 57% and 9/11, 82%, respectively). However, embryo development rates were lower (P<0.05) when frozen spermatozoa were placed within the oviduct (1/12, 8%). In Experiment II, embryo development rates were higher (P<0.05) when cooled semen was used for intrauterine (19/23, 83%) versus intraoviductal (4/16, 25%) inseminations. We concluded that intraoviductal insemination can be successfully performed using fresh spermatozoa. However, the use of cooled and frozen spermatozoa for intraoviductal inseminations was less successful, and needs further investigation.     

Cervical versus intrauterine insemination of ewes using fresh or frozen semen diluted with aloe vera gel

This study was conducted at Belen de Escobar, Argentina, in March and April 1987. Experimental work on synchronization of estrus, deep-freeze conservation of ram semen and small fertility trials involving cervical and intrauterine (i.u.) insemination methods was undertaken. A total of 80 Corriedale ewes were used in seven insemination trials. Insemination trials were grouped into two experimental groups for comparison of 1) frozen semen diluted with an experimental extender and a control diluent inseminated cervically or i.u. in synchronized/superovulated ewes and 2) cervical insemination of fresh diluted or frozen semen in ewes inseminated at natural estrus or in ewes that were synchronized/superovulated. An overall ovulation rate of 8.7 +/- 0.5 was obtained by using a superovulatory regimen consisting of 3 mg Norgestomet implants and a total dose of 18 mg follicle stimulating hormone-pituitary (FSH-P). Numbers of ova recovered per ewe following superovulation ranged from 4.3 to 5.4. In experimental Group I, fertilization rates improved when laparoscopic intrauterine AI was used compared with cervical insemination (P<0.05). Fertility rates of i.u. and cervical insemination of frozen semen diluted with the experimental extender showed satisfactory fertilizing capacity. In experimental Group II, a lower number of fertilized ova were recovered from ewes inseminated with frozen semen (P<0.02), irrespective of their estrus manipulation.     

Successful uterine insemination of fallow deer with fresh and frozen semen

Six fallow does were inseminated directly into the uterine horns 72 h (three does) or 78 h (three does) after the removal of progestagen intravaginal sponges. Three does were inseminated with fresh (two at 72 h and one at 78 h) or frozen-thawed (one at 72 h and two at 78 h) semen. The semen used had been collected by electroejaculation and had been stored for 2 yr in liquid nitrogen in a Tris, citric acid, glycerol diluent containing 2.25% egg yolk. Three does each produced a live fawn to insemination and all does had been inseminated 72 h after removal of sponges; two with fresh semen and one with frozen semen. The remaining three does failed to conceive to insemination, but did produce fawns to mating at a subsequent estrus.     

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen-thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5+/-5.4 x 10(6) sperm (range, 6.8-22 x 10(6)) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 x 10(6) sperm, with 70+/-5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17beta and progesterone were determined in most queens on the day of AI and again 30-40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P=0.58); overall 33% (5/15) of the queens became pregnant. For frozen-thawed semen, AI was consistently done 28h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P=0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen-thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.     

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen–thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5 ± 5.4 × 10⁶ sperm (range, 6.8–22 × 10⁶) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 × 10⁶ sperm, with 70 ± 5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17β and progesterone were determined in most queens on the day of AI and again 30–40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P = 0.58); overall 33% (5/15) of the queens became pregnant. For frozen–thawed semen, AI was consistently done 28 h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P = 0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen–thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.     

Hormonal and behavioural detection of oestrus in blackbuck, Antilope cervicapra, and successful artificial insemination with fresh and frozen semen

Hormonal detection of urinary pregnanediol-3 alpha-glucuronide proved an effective method of monitoring the progress of oestrous cycles in the blackbuck; observation of sexual behaviour in a vasectomized male was, however, a more practical procedure. Good correlation was observed between the occurrence of minimal pregnanediol concentrations in females and the maximal behavioural response by the male. On the basis of intervals between periods of behaviourally detected oestrus, a mean cycle length of 16.9 days (+/- 0.62, s.e.m.) was derived from 12 cycles (4 animals). Eleven females were inseminated in this study, 8 with freshly collected semen and 3 with frozen semen; 6 calves were obtained, 1 after the use of frozen semen. Pregnancy was monitored by measurements of pregnanediol-3 alpha-glucuronide excretion and by ultrasound scanning. The mean interval between insemination and parturition was 183.3 days inclusive, ranging from 182 to 186 days.     

Lambing rates and litter sizes following intrauterine or cervical insemination of frozen/thawed semen with or without oxytocin administration

Intrauterine insemination by laparoscopy is required to achieve acceptable lambing rates in ewes when using frozen semen but the procedure has evoked welfare concerns. Oxytocin has been used to dilate the cervix as a means of accessing the uterus during conventional cervical insemination, but its effect on fertility is not well documented. Three hundred crossbred ewes were synchronised in estrus and randomly allocated to one of three insemination procedures using frozen/thawed semen containing 400 x 10(6)/ml progressively motile sperm: single cervical (0.2 ml), multiple cervical (4 x 0.05 ml) or laparoscopic (0.05 ml per uterine horn). The effects of each insemination procedure on lambing rate (percentage of treated ewes lambing) and litter size (lambs per ewe lambing) were tested with and without oxytocin (10 IU given i.m.) prior to fixed-time insemination. Oxytocin did not permit complete cervical penetration in any ewes and neither lambing rate nor litter size was influenced by the number of inseminations. Lambing percentages were 69 and 42 (P < 0.01) for the laparoscopic and cervical insemination methods, respectively, and oxytocin reduced these to 58 (NS) and 10 (P < 0.001) percent, respectively. Corresponding litter sizes for ewes not receiving oxytocin were 1.91 and 1.51 and for those receiving oxytocin, 1.83 and 1.41 (laparoscopic versus cervical, P < 0.02). Thus, in the absence of complete cervical penetration at insemination, 10 IU oxytocin decreased the number of ewes lambing but had no effect on their litter size.     

Comparison of fertility results after vaginal insemination using different thawing procedures and packages for frozen ram semen

Background  

The effect of different thawing procedures for ram semen frozen in minitubes and mini straws on the fertility of sheep was tested in a field trial.     

Fertility of ram semen frozen in Bioexcell and used for cervical artificial insemination

The current use of ingredients of animal origin, such as egg yolk, in semen extenders presents a risk of microbial contamination, and has led to the search for alternatives. Such an extender is commercially available for bull semen (Bioexcell), IMV, L'Aigle, France), and it has previously been tested in vitro for freezing ram semen, with satisfactory results. The aim of the present study was to compare the fertility results of ewes in Uruguay, after cervical insemination with ram semen that was frozen in Bioexcell versus semen frozen in a conventional milk-egg yolk extender (control). Semen from five Corriedale rams was frozen, using a split sample design, in either milk-egg yolk or Bioexcell extender, using a two-step extension method. The sperm parameters assessed after thawing were subjective motility, membrane integrity (SYBR-14/PI), and capacitation status (CTC). Thawed semen was inseminated intracervically once during spontaneous estrus in 970 Corriedale ewes that grazed in natural pastures, under extensive management conditions. Fertility was recorded as nonreturn rates at 21 days (NRR-21) and 36 days (NRR-36) after artificial insemination (AI), as well as pregnancy rate (PR-US, diagnosed ultrasonographically 50 days after AI of the last ewe). Subjective motility was slightly higher in Bioexcell than in the milk extender (47 vs. 46.5%; NS), as was membrane integrity (38 vs. 37.7%; NS) and the percentage of uncapacitated spermatozoa (28.5 vs. 26.3%; NS). There were no statistically significant differences in fertility rates found between Bioexcell and the control extender: NRR-21 (35.9 vs. 33.2%), NRR-36 (34.8 vs. 32.6%), and PR-US (28.4 vs. 27.2%). In conclusion, Bioexcell appears to be an alternative to the conventional milk-egg yolk extender for freezing ram semen, and provides similar fertility results after cervical AI under extensive management conditions. Thus, Bioexcell, containing no additives of animal origin, can offer a safer alternative when frozen semen is used for introducing new genetic material into a flock or a country.     

Artificial insemination with frozen,thawed semen and pregnancy diagnosis in addax (Addax nasomaculatus)

Nonsurgical artificial insemination (AI) procedures were performed using frozen, thawed semen in two adult female addax. Pregnant mares' serum gonadotropin (PMSG) (300 IU) and prostaglandin (PG) (125 μg) was administered intramuscularly to female 1 on days ?3 and ?1, respectively, where day 1 was the first day of AI. Only one injection of PG was used, as a low serum progesterone (PROG) concentration (0.5 ng/ml) and a low pregnanediol glucuronide (PdG) concentration (0.36 ng PdG/mg creatinine) had been recorded on day ?13. A total of 219 × 10⁶ motile sperm was deposited in the os cervix or anterior vagina on days 1–4. High serum PROG levels (9.4–15.8 ng/ml) and high urinary PdG levels (1.35–1.63 ng PdG/mg creatinine) were observed on days 23, 31, and 39, and the fetal heart was seen beating during an ultrasound exam on day 45. A male calf was born on day 266, 8.7 mo after insemination. This is the first live birth resulting from AI of an antelope with frozen, thawed semen. The Synchro-mate-B system of estrous control was used in female 2. The implant was inserted, and the injection was given on day ?11 per the manufacturer's instructions. The implant was removed on day ?1, and a total of 220 × 10⁶ motile sperm was deposited in the os cervix or anterior vagina on days 1 and 2. High serum PROG levels (17.5–24.5 ng/ml) were observed on days 13, 21, and 29. Although the fetal heartbeat was observed during an ultrasound examinataion on day 42, the fetus was not carried to term. Details of the radioimmunoassay used to determine the luteinizing hormone immunoreactivity of urine samples are presented.     

Successful artificial insemination in the corn snake,Elaphe gutatta,using fresh and cooled semen

The design and implementation of assisted reproductive technology to improve genetic diversity and augment captive populations is an important but rarely applied research field in reptiles. Using the corn snake (Elaphe gutatta) as a model, the Henry Doorly Zoo recently produced offspring born as a result of artificial insemination using both fresh, diluted semen, and diluted semen stored at refrigeration for 3 days. Semen was collected noninvasively from sexually mature male corn snakes using a gentle massaging technique, extended in medium then inseminated into the oviducts of adult females. Using molecular genetic techniques to confirm or refute the success of the insemination using primers developed for the black rat snake, Elaphe obsolete, all possible parents and offspring genotypes were evaluated. A paternity‐by‐exclusion analysis verified that the offspring were in fact a result of artificial insemination. Zoo Biol 26:363–369, 2007. © 2007 Wiley‐Liss, Inc.     

Fertilized eggs obtained from transplantation of frozen ovaries and parthenogenesis in combination with artificial insemination of frozen semen of the silkworm,Bombyx mori

A reliable method is reported for the long-term preservation of ovaries and spermatozoa of the silkworm (Bombyx mori). Three studies are presented. In the first, ovaries were removed from larvae at either 3rd, 4th, or 5th instar, cryopreserved, and stored in liquid nitrogen. Thawed ovaries were transplanted to surgically castrated female larvae at the same or a different developmental stage. The highest percentage of recipient females producing eggs resulted into either 3rd or 4th instar larvae (respectively, 22.1 and 8.7%). Similarly, the highest levels of other measurements of successful cryopreservation and transplanted ovary, and number of eggs laid, occurred with the same combination of donor and recipient developmental stages. Other combinations of ovary/recipient developmental stages yielded lower results. In the second experiment, semen was collected from male moths, cryopreserved, and then thawed semen was diluted with trypsin solution and artificially inseminated into females obtained from the best conditions of first experiment. A small percentage of inseminated moths laid eggs (8-10.3%) compared to that of controls (100%). In addition, the fertility of eggs from experimental moths was lower than that of control females (respectively, 40.3-88% and 97.5%). In the third experiment, eggs were surgically removed from ovarian tubules of moth following transplantation of thawed ovaries and subjected to parthenogenetic activation and artificial hatching. As expected, all resulting moths were female and, following natural mating or artificial insemination with thawed semen, yielded normal offspring at high rates.     

Effect of an asynchrony between ovulation and insemination on the results obtained after insemination with fresh or frozen sperm in rabbits

The effects of the introduction of an 8-h asynchrony between ovulation and insemination on litter size components from rabbits were assessed. A total of 202 females belonging to a maternal line were used. Fresh and frozen sperm were used to perform the inseminations. Sperm was frozen with an extender composed of 1.75 M DMSO and 0.05 M sucrose. Four experimental groups were obtained depending on the type of sperm used (fresh or frozen) and on the moment that ovulation had been induced relative to the insemination (at the same time as insemination (t(0)) or 8 h before insemination (t(8))). Laparoscopy was performed on 12th day of pregnancy in pregnant females, and the ovulation rate, normal and total implanted embryos were noted. At kindling, total and live-born rabbits were noted. Results showed that better results were obtained after insemination with fresh semen than with frozen sperm (for females in the group t(0): 79% versus 61% fertility rate, 10.2 versus 6.4 normal implanted embryos and 8.1 versus 5.2 total number born, for fresh and frozen sperm, respectively). On the other hand, after the introduction of an 8-h asynchrony between ovulation and insemination, results were lower for both fresh (50% fertility rate, 7.5 normal implanted embryos and 5.7 total number born for the group of the asynchrony) and frozen sperm (31% fertility rate, 4.6 normal implanted embryos and 3.4 total number born for the group of the asynchrony). Although an approach between the moment of insemination and ovulation is justified when sperm survival could be compromised, results observed after the induction of an 8-h asynchrony were not those expected, perhaps due to the ageing of the oocytes before being fertilised, leading to both lack of fertilisation or early embryonic mortality.     

Effects of added caffeine on results following artificial insemination with fresh and refrigerated rabbit semen

Lactating rabbits (n=1335) were artificially inseminated to study the effect of the addition of caffeine on rabbit semen stored for up to 96 h. Concentration of 0-5 mM/l were tested. Whereas a concentration of 0.2 mM/l increased spermatozoa motility, higher concentration values adversely affected reproductive parameters. Spermatozoa stored at 18 degrees C for 72-96 h did not have the capacity to react with caffeine when it was added before insemination. Caffeine did not enhance fertility or prolificacy, regardless of its ability to increase sperm motility.     

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

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