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].

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 present study investigated effects of three semen extenders and storage temperatures on post-thaw characteristics of Bryde's whale spermatozoa. Spermatozoa were collected from the vasa deferens of three mature Bryde's whales captured during the Japanese whale research in the north-west Pacific (May to August 2007) after death. The three semen extenders used for freezing were 1) a commercialized synthetic extender (AndroMed: AM), 2) Trisbased + 10% bovine serum albumin (BSA) and 3) Tris-based + egg yolk (EY). The sperm samples from the three whales were frozen with the three extenders, and the post-thaw spermatozoa were stored at three different temperatures (35 C; 20-25 C, room temperature; and 5 C) for 0, 6, 12, 24, 48 and 96 h. At each time-point, total and progressive motility (PM), viability (live or dead), the hypo-osmotic test, defective acrosomes and malformation were examined. Immediately after thawing, AM resulted in similar recovery rates (60.4 and 83.3%) in 2 of the 3 whales examined and had comparable post-thaw recovery rates to those obtained using the EY and BSA extenders. Immediately after thawing, the proportion of PM in EY (17.6%) was higher (P<0.05) than that in BSA (15.0%). In the hypo-osmotic test, the proportions of AM (26.0%) and BSA (25.2%) were higher (P<0.05) than that of EY (17.3 %). The three extenders had similar viabilities (36.7, 37.9 and 32.1%, respectively), but the viability of BSA was higher (P<0.05) than that of EY. The present study showed that a synthetic semen extender, AndroMed, could be used for cryopreservation of whale spermatozoa in addition to Tris-based extenders containing bovine serum albumin or egg yolk. Storage of the post-thaw Bryde's whale spermatozoa was better at 5 C than at room temperature or 35 C. The frozen-thawed Bryde's whale spermatozoa maintained their motility and viability for at least two days at room temperature and for four days at 5 C. [5] used Bryde's (Balaenoptera edeni) whale spermatozoa freshly-diluted with m-PBS and stored at 4 C for in vitro fertillization (IVF) in the field. However, it has not been demonstrated yet how long the viability of frozen-thawed spermatozoa is maintained and what the most appropriate temperature is for post-thaw storage in baleen whales, including Bryde's whales. For domestic animals including sheep, post-thaw semen is usually maintained at 37 C for examinations of their motility and viability, but the body temperatures (35.5 to 37 C) of dolphins and whales are lower than those of other mammals, and no one has measured the body temperatures of live baleen whales, including Antarctic minke and Bryde's whales.There has been recent movement to eliminate all animal ingredients, such as egg yolk, milk and even bovine serum albumin (BSA), from semen extenders. Our recent studies [6-8] on artificial insemination (AI) in sheep have clearly shown that a soybean-based semen extender (AndroMed) is useful and comparable to the conventional semen extenders containing egg yolk or BSA. Fukui et al. [6] compared ...