Abstract. In this study, two following experiments were performed to improve post-thaw motility and viability of frozen-thawed ram spermatozoa. We examined i) the effects of different concentrations of bovine serum albumin (0, 0.3, 1, 5, 10 and 15% BSA) in semen diluents lacking egg yolk and ii) the effects of four semen diluents, fructose (F: control) and trehalose (T) in semen diluents containing egg yolk, 15% BSA in semen diluents without egg yolk (BSA), and modified phosphate buffered saline (m-PBS). Frozen-thawed spermatozoa were examined for progressive sperm motility, viability, morphological abnormality, sperm tail swelling test, and sperm acrosome integrity. In Experiment 1, the rates of sperm motility immediately after thawing (0 h) were significantly (P<0.05) higher in the 10 and 15% BSA groups (55.0 ± 2.9 and 58.3 ± 6.7%, respectively) than in the positive control (F) group (41.7 ± 4.4%). The rate of sperm viability in the negative control (0% BSA) group (80.2 ± 3.3%) was significantly (P<0.05) lower than in the positive control (F) group (89.8 ± 1.5%), but when compared with the F group, no significant differences were found among the 0.3, 1, 5, 10 and 15% BSA groups at 0 h. The rates of sperm morphological abnormality of the 10 and 15% BSA groups (6.5 ± 1.3 and 6.3 ± 1.1%, respectively) were significantly (P<0.05) lower at 0 h than that in the 1% BSA group (16.3 ± 5.2%). In Experiment 2, T addition improved (P<0.05) the post-thaw motility compared with the F and BSA groups. Furthermore, at 3 and 6 h, the post-thaw motility of the T group (36.3 ± 2.4 and 25.0 ± 2.0%, respectively) was significantly (P<0.05) higher than in the BSA (26.3 ± 2.4 and 18.8 ± 1.3%, respectively) and F (28.8 ± 3.8 and 18.8 ± 2.4%, respectively) groups. The post-thaw sperm motility and viability in the m-PBS group were significantly (P<0.05) lower than those of the control (F), T, and BSA groups throughout all observation points. These results indicate that 10 and 15% BSA can be substituted for egg-yolk for ram semen diluent and that the addition of trehalose enhances motility and viability of ram spermatozoa after freezing and thawing. Key words: Bovine serum albumin (BSA), Semen diluent, Sheep, Spermatozoa, Trehalose (J. Reprod. Dev. 52: [675][676][677][678][679][680][681][682][683] 2006) t has been reported that frozen-thawed spermatozoa are greatly damaged during the freezing and thawing processes [1][2][3][4][5]. This damage may decrease sperm motility and the fertilization rate after artificial insemination (AI). Several semen analysis methods have been developed to evaluate the functional parameters of spermatozoa more objectively. The hypo-osmotic swelling test (HOS-test) has been applied to various species, such as dogs [6,7], cattle [8,9], horses [10][11][12], rams [13], minke whales (Balaenoptera bonarensis) [13] and humans [14,15]. It has been recognized that the HOS-Test is a simple and useful method with good reliability and repeatability for examination of the sperm plasma membrane.

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.Two experiments were conducted to compare the effect of estrus induction by controlled internal drug release (CIDR) and intravaginal cream containing 500 mg progesterone (P cream) in ewes during the non-breeding season. In the first experiment, twenty-four ewes were randomly grouped for two treatments with the different intravaginal devices for 12 days: Group A was the CIDR group and Group B was the P cream group. Blood was collected from all treated ewes, and progesterone (P4), estradiol 17-β (E2) and luteinizing hormone (LH) concentrations were measured by enzyme immunoassay. In the second experiment, the conception rates from natural mating, estrusdetected AI (inseminated 12 h after estrus detection), or fixed-time AI (inseminated 42 h after removal of an intravaginal device) in 127 ewes treated with CIDR or P cream were compared. In Experiment 1, the rate of estrus induction and the time of estrus onset after device removal were 91.7% and 36.3 ± 15.7 h in Group A, and 100% and 35.0 ± 12.6 h in Group B, respectively. There were no significant differences between the devices. The mean plasma P4 concentration in Group B was significantly (P<0.01) lower than Group A between day -9 and day -1 (Day 0: the day of device removal). However, no significant differences were found in the mean E2 concentrations of the two groups after treatment. The mean time of estrus onset in ewes with an observed LH surge and the time of LH surge after treatment were 23.3 ± 8.7 h and 30.3 ± 5.0 h for Group A and 27.6 ± 6.5 and 26.3 ± 8.0 h for Group B, respectively, and there were no significant differences. However, a significant difference (P<0.05) was found in the mean time from the time of estrus onset to LH surge between Group A (6.4 ± 6.7 h) and Group B (-1.3 ± 4.1 h). In Experiment 2, the conception rates for natural mating, estrus-detected AI, and fixed-time AI were 55.0, 29.4, and 25.0% for Group A and 40.7, 25.0, and 42.1% for Group B, respectively, and there were no significant differences. These results suggest that the effect of induction of estrus and ovulation and the rate of conception after treatment were comparable to CIDR even though the plasma P4 concentration of the P cream method tended to be low during the insertion period. Key words: Estrus induction, Fertility, Intravaginal device, Non-breeding season, Sheep (J. Reprod. Dev. 51: [805][806][807][808][809][810][811][812] 2005) ethods of estrus induction in ewes using various intravaginal devices impregnated with progesterone or synthetic progestogen have been developed and used throughout the world.The main method is an intravaginal sponge

Abstract. The aim of the present study was to compare three methods of estrus synchronization in ewes during the non-breeding season. Forty-two ewes were randomly grouped for three treatments with different intravaginal devices for 12 days: Group A) CIDR, Group B) Self-made P sponge, Group C) MAP (medroxyprogesterone acetate) cream sponge. Furthermore, all groups were divided into two treatments with (R) or without ram presence to examine the "ram effect". Blood was collected from all treated ewes, and progesterone (P4), estradiol 17-β (E2) and luteinizing hormone (LH) concentrations were measured by enzyme-immnoassay. All ewes showed estrus behavior between Day 0 to 3 after device removal, and the mean onset times of their estrus were 23.0, 33.0 and 21.0 h for Groups AR, BR and CR, respectively. On Day 5 as examined by laparoscopy, the ovulation rates (and number of ovulated ewes) were 1.45 (11/11), 1.25 (12/14) and 1.21 (14/14) for Groups A, B and C, respectively. In Group C, the time to LH surge was significantly (P<0.05) later (32.4 h) than those in Groups A (27.0 h) and B (25.5 h). Ram presence did not affect the number of ovulated ewes, ovulation rate or time to LH surge. The ram introduction group had significantly (P<0.05) lower E2 concentrations during the period from 0 h to 36 h than the groups without ram presence. These results suggest that the self-made P sponge or MAP cream sponge was effective as well as CIDR, and ram introduction was not necessary, for induction of estrus and ovulation during the non-breeding season. Key words: Induction of estrus, Intravaginal device, Non-breeding season, Sheep (J. Reprod. Dev. 50: [63][64][65][66][67][68][69] 2004) strus synchronization is generally applied for reproductive management of sheep flocks worldwide [1], and several methods have been performed with varying degrees of success [2]. The attempted methods are control of daily length and/ o r h o r m o n a l t r e a t m e n t s s u c h a s n a t u r a l p r o g e st e r o n e , s y n t h e t i c p r o g e s t o g e n s ,