Abstract. The present study aimed to compare the fertility of ewes intrauterinally inseminated with frozen-thawed semen using a soybean-based semen extender (AndroMed) with those of ewes intrauterinally inseminated with frozenthawed semen using a Tris-based extender containing either egg yolk or BSA. Suffolk ewes (n=104) were treated with an intravaginal sponge containing 40 mg fluoroprogesterone acetate (FGA) for 12 days and an intramuscular injection of 500 IU equine chorionic gonadotropin to induce estrus and ovulation during the non-breeding season (July, 2007). Intrauterine insemination was carried out 40-46 h after removal of the FGA sponge (n=90), regardless of the incidence of estrus. The pregnancy rates were not significantly different among the semen extenders containing egg yolk (64.5%) or BSA (58.6%) and AndroMed extender (56.7%). The lambing rates (64.5, 55.2 and 56.7% for the semen extenders containing egg yolk, BSA and AndroMed, respectively) and prolificacy (1.59 to 1.75) were also not significantly different. The present results indicate that an egg yolk-containing semen extender can be replaced with the non-animal derived extender AndroMed, which could be used for intrauterine insemination using frozen-thawed ram semen without reducing fertility. Key words: Artificial insemination, Fertility, Semen extender, Sheep (J. Reprod. Dev. 54: [286][287][288][289] 2008) gg yolk-based semen extenders have been widely utilized for cryopreservation of semen from farm animals including sheep [1][2][3]. However, it is not always easy to prepare semen extenders consistent with quality standards because of the individual quality differences inherent in egg yolk due to the numbers of days after laying and the storage period. Also, addition of egg yolk reduces the acrosome integrity of goat spermatozoa [4], and high egg yolk concentrations reduce the post-thawing viability of ejaculated spermatozoa in several species, such as goats [5], rams [1] and water buffaloes [6]. Furthermore, there has been movement recently to eliminate all animal ingredients, including egg yolk, milk or bovine serum albumin (BSA), in order to design a defined semen extender. Removal of chicken egg yolk from semen extenders would provide several advantages, such as improved consistency in the components of semen extenders and elimination of hygienic risks. Therefore, development of a synthetic semen extender free of animal sources has been desired. A soybean lecithin-based extender (AndroMed; Minitub, Tiefenbach, Germany) has been developed and utilized for bovine [7][8][9] and mountain gazelle semen [10]. Fresh and frozen ram semen diluted with a synthetic semen extender, AndroMed, has been inseminated with satisfactory fertility results in Norway (Paulenz H.: personal communication).Low fertility (20-30%) in ewes inseminated with frozen semen into the cervical orifice, an ordinal deposition site for artificial insemination (AI) in sheep, has not been applied fully on the field, except for intrauterine AI using laparoscopy (60-80% in...

Abstract. The present study was conducted to examine the fertility of ewes inseminated intrauterinally with frozen semen using semen extender containing either egg yolk or bovine serum albumin (BSA). Sixty Suffolk and cross-bred ewes were treated with controlled internal drug release (CIDR) devices during the non-breeding season (July 2006). A CIDR was inserted into the vagina for 12 days and an intramuscular injection of 500 IU equine chorionic gonadotropin was administered one day before its removal. Ejaculates from a suffolk ram were diluted with a Tris-based extender containing either 15% (v/v) egg yolk or 10% (w/v) BSA, and the diluted semen was frozen in 0.25 ml straws. A fixed-time intrauterine artificial insemination (AI) was performed 43-47 h after CIDR removal, regardless of incidence of estrus. There was no significant difference in pregnancy rates at 60 days after AI between the extenders containing egg yolk (66.7%, 20/30 animals) or BSA (65.5%, 19/29 animals). Furthermore, there were no significant difference in the lambing rates (66.7% and 62.1%) and prolificacy (1.25 and 1.56) between the two semen extenders. The present study indicates that a semi-defined semen extender containing 10% BSA produces fertility after intrauterine AI that is similar to that achieved with semen extender containing egg yolk. o s t s e m e n e x t e n d e r s f o r f r e e z i n g o f spermatozoa, including ram semen contain egg yolk, milk, or a combination of the two as a basic ingredient. Although egg yolk is beneficial for sperm cryopreservation because it protects a g a i n s t c o l d s h o c k [ 1 ] , t h e r e a r e s e v e r a l disadvantages of addition of egg yolk to semen extender. Preparation of uniform semen extenders containing egg yolk is difficult because individual egg yolk quality may vary depending on the number of days after laying and the storage period. Furthermore, addition of egg yolk reduces the acrosome integrity of goat spermatozoa [2], and high egg yolk concentrations reduce the postthawing viability of ejaculated spermatozoa in several species, such as goats [3], rams [4] and water buffaloes [5]. Finally, development of a chemically defined semen extender would be required in order to eliminate possible infectious origins from egg yolk added to the semen extender. Several investigations have been conducted for development of semen extenders that do not contain egg yolk, such as a defined extender containing soybean lecithin for bovine and wild gazelle semen [6][7][8][9][10], and bovine serum albumin (BSA) has also been used as a substitute of egg yolk for rainbow trout and turkey spermatozoa [11,12].

In this study, two successive field trials were conducted during the non-breeding season to investigate various factors affecting on fertility of Suffolk ewes after intrauterine insemination with frozen-thawed semen. In the first year (Experiment 1), three sperm numbers per insemination dose (0.25, 0.5 and 1 million sperm) and five sheep farms were used, and in the second year (Experiment 2), parity, age, body weight, body condition score (BCS) and postpartum days were investigated to compare pregnancy and lambing rates. High pregnancy and lambing rates (70.6 and 70.6%, respectively) were obtained with 0.25 million sperm per dose. There were no significant differences in the pregnancy and lambing rates among the five farms, but there was a tendency for one farm to have higher pregnancy (75.8%, P=0.065) and lambing (72.7%, P=0.077) rates than those (46.7-53.3% and 45.2-53.3% for the pregnancy and lambing rates, respectively) of the other farms. In Experiment 2, ewe age significantly affected both the pregnancy and lambing rates. Nulliparous ewes had a higher lambing rate (72.0%) than that (44.2%) of multiparous ewes, but a significant difference was not revealed. Regardless of body weight, BCS tended to be an important factor influencing on fertility of ewes. Body weight and the postpartum days did not affect the fertility of ewes. It was concluded from these results that the fertility of Suffolk ewes after intrauterine insemination with frozen semen was significantly influenced by sperm number per dose and ewe age. Nulliparous ewes at less than three years of age and with a BCS of more than 3.0 are expected to have higher fertility than other ewes.

Abstract. 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 nonbreeding 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 frozenthawed (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 freshdiluted (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. Key words: Artificial insemination (AI), Fertility, Semen extender, Sheep (J. Reprod. Dev. 55: [50][51][52][53][54] 2009) n artificial insemination (AI) of farm animals including sheep, egg yolk or milk based semen extender have been widely utilized for semen cryopreservation [1][2][3]. Our pervious studies [4,5] reported that bovine serum albumin (BSA) could be used instead of egg yolk as a semen extender. However, egg yolk and BSA are of ani...

ABSTRACT. The selection of sheep with scrapie-resistant PrP genotypes is one of the control measures for transmissible spongiform encephalopathies in ruminants. In this study, we investigated the frequencies of PrP genotypes in meat breeds in Japan. The nationwide surveillance revealed that nearly half of the Suffolk sheep, a major meat breed in Japan, carried scrapie-susceptible AQ/AQ and AQ/VQ genotypes. In addition, the VQ haplotype, which confers high susceptibility to scrapie within sheep, was also found in Poll Dorset sheep. A trial of selective breeding using sires with scrapie-resistant PrP genotypes AQ/AR and AR/AR could raise the ratio of scrapie-resistant sheep from less than 50% to 80% within 3 years. However, the use of sires with the AR/AR genotype and the selection of ewes would be required to achieve a higher ratio of scrapie-resistant sheep. KEY WORDS: prion, scrapie, transmissible spongiform encephalopathy.

Artificial insemination, especially with the use of frozen semen, is one of the important tools for embryo transfer program in sheep. The present study investigated the effects of insemination times, breeds, and two inseminators on the fertility of ewes intrauterinally inseminated with frozen–thawed ram semen imported from New Zealand. At 8 sheep farms located in Hokkaido, Japan, during the breeding season (October to December) in 2005, a total of 64 mature (1- to 6-year old) Suffolk (32 heads) and Polled Dorset (32 heads) ewes were used. The ewes were treated with controlled intravaginal drug release (CIDR containing 0.3 g progesterone; Pharmacia & Upjohn, Ltd., Auckland, New Zealand) for 12 days and an injection of 500 IU equine chorionic gonadotropin one day before CIDR removal. The fixed-time intrauterine inseminations (early: 43–46 h; late: 47–50 h) after CIDR removal were performed using the frozen–thawed semen from a Suffolk and Polled Dorset ram by two inseminators. The effects of breeds (Suffolk and Polled Dorset), fixed-time insemination times (early and late phases), and two inseminators on pregnancy (number of pregnant ewes/number of ewes inseminated, 60 days after insemination) and lambing (number of lambed ewes/number of ewes inseminated) rates were analyzed by chi-square test. The prolificacy was compared by Student's t-test, and differences were also analyzed by Tukey's omega procedure. The effect of the different farms on fertility was not examined due to the small numbers of ewes per farm. Pregnancy (60.0 and 72.4%, respectively) and lambing (60.0 and 71.4%, respectively) rates were not significantly different between Suffolk and Polled Dorset ewes. The inseminators also did not affect pregnancy (62.6 and 68.8%) and lambing (62.6 and 67.7%) rates. For the insemination times, the lambing rate tended to be higher (P ≤ 0.10) in the early insemination than in the late insemination (76.7% and 53.6%, respectively). The present results show acceptable fertility in ewes inseminated with Suffolk and Polled Dorset frozen semen imported from New Zealand. The early intrauterine insemination (43–46 h after CIDR removal) tended to result in higher fertility than the late insemination (47–50 h after CIDR removal). From 38 lambed ewes, 60 newborn lambs were produced, and this has provided new blood lines of Suffolk and Polled Dorset sheep in Japan.

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