Fertility after artificial insemination using a soybean-based semen extender in sheep

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 frozen-thawed 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.     

Fertility of ewes inseminated intrauterinally with frozen semen using extender containing bovine serum albumin

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.     

Follicular Dynamics,Interval to Ovulation and Fertility After AI in Short‐Term Progesterone and PGF2α Oestrous Synchronization Protocol in Sheep

The study was aimed to assess the influence that short‐term progesterone treatments have on follicular dynamics, oestrus and ovulation in sheep. The treatment was tested thereafter in a field trial to assess its fertility after AI with fresh semen. In a first experiment, 12 ewes without CL were grouped to receive a new (n = 6) or used CIDR (n = 6) for 7 days and blood samples were obtained to follow plasma progesterone profiles. In a second experiment, 39 cycling ewes were synchronized by a 7‐day P4+PGF2α protocol using a new (n = 20) or a 7‐day used CIDR (n = 19). Half of both groups received 400 IU eCG and half remained untreated as controls. Ultrasound ovarian examination and oestrous detection were used to compare follicular dynamics, oestrus and ovulation in both groups. In a third experiment, 288 ewes in 3 farms were synchronized by the short‐term P4+PGF2α+eCG protocol and ewes were AI with fresh semen 24 h after oestrous detection. Lambing performance was used to test the fertility of the treatment. In Experiment 1, ewes with new inserts presented higher P4 concentration than ewes with used inserts throughout the sampling period (p < 0.05) and exhibited a P4 peak at days 1‐2 of the treatment that was not observed in ewes with used inserts. In Experiment 2, ewes treated with new and used inserts show similar ovarian and behavioral traits (p > 0.10). However, ewes treated with eCG show shorter interval to oestrus (p = 0.004) and tend to have larger mature CL (p = 0.06). In Experiment 3, oestrous presentation and lambing performance after AI with fresh semen was considered normal compared to published results. Results suggest that the oestrous synchronization protocol based on P4+PGF2α allows little control of follicular dynamics without compromising fertility after AI with fresh semen provided that eCG is added at the end of the treatment.     

Comparison of estrus induction and subsequent fertility with two different intravaginal devices in ewes during the non-breeding season

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 (P(4)), estradiol 17-beta (E(2)) and luteinizing hormone (LH) concentrations were measured by enzyme immunoassay. In the second experiment, the conception rates from natural mating, estrus-detected 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 P(4) 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 E(2) 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 P(4) concentration of the P cream method tended to be low during the insertion period.     

冷冻精液输精深度和时间对蒙古羊受胎率的影响

[目的] 研究冷冻精液输精深度和时间对蒙古羊受胎率的影响。[方法] 选取体况健康、繁殖机能正常的蒙古羊母羊作为试验动物,采用孕酮海绵栓对受体母羊进行同期发情处理。应用常规人工输精法（n=135）、过子宫颈输精法[过子宫颈第一皱褶（1~2 cm）输精（n=120）,过子宫颈第二到第三皱褶（3~4 cm）输精（n=120）]、腹腔镜辅助子宫内输精法（n=128）对发情后的母羊进行冷冻精液人工授精。接受常规人工输精法和过子宫颈输精法处理的受体母羊在发情后约14 h和22 h各输精1次,接受腹腔镜辅助子宫内输精法处理的受体母羊在发情后30~36 h输精1次。输精后40 d用B超仪检测母羊妊娠情况,计算并比较接受不同输精方法处理母羊的受胎率。[结果] 腹腔镜辅助子宫内输精法的受体母羊受胎率平均值最高,为60.2%;其次为过子宫颈第二到第三皱褶（3~4 cm）输精,受胎率平均值为58.3%;过子宫颈第一皱褶（1~2 cm）输精和常规输精法的受体母羊受胎率平均值分别为37.5%和36.3%。统计学分析表明,腹腔镜辅助子宫内输精法和过子宫颈第二到第三皱褶（3~4 cm）输精的受体母羊受胎率平均值显著（P<0.05）高于常规输精法和过子宫颈第一皱褶（1~2 cm）输精。[结论] 蒙古羊母羊发情后30~36 h采用腹腔镜辅助子宫内输精法完全穿刺子宫颈输精1次,可获得比常规输精法更高的受胎率;采用过子宫颈输精法在母羊发情后约14 h和22 h各输精1次,当授精深度接近4 cm时可达到与腹腔镜辅助子宫内输精法接近的受胎率。     

Autologous Whole Ram Seminal Plasma and its Vesicle-free Fraction Improve Motility Characteristics and Membrane Status but not In Vivo Fertility of Frozen–Thawed Ram Spermatozoa

Motility characteristics (assessed subjectively and with computer-assisted semen analysis) and membrane status (after staining with chlortetracycline) of washed and non-washed frozen-thawed ram spermatozoa were evaluated after incubation in buffer and buffer containing autologous whole seminal plasma or one of its two fractions: the pellet of membrane vesicles obtained by ultracentrifugation (and used at three times normal protein concentration) or the vesicle-free supernatant fraction. Whole seminal plasma and supernatant, but not membrane vesicles, improved the motility characteristics of spermatozoa after 3 and 6 h of post-thaw incubation compared with the control buffer. Resuspension and incubation with whole seminal plasma, supernatant or membrane vesicles lowered the proportion of acrosome-reacted frozen-thawed spermatozoa compared with the control buffer. Unwashed frozen-thawed semen from three rams, incubated with autologous whole seminal plasma or its fractions and inseminated using cervical or intrauterine artificial insemination, had no effect on pregnancy rates of ewes in synchronized oestrus. However, fertility was higher after laparoscopic than cervical insemination (44.9 vs 12.3%, p < 0.001). In conclusion, resuspension and incubation of frozen-thawed ram spermatozoa in autologous whole seminal plasma or its vesicle-free supernatant fraction improved their motility characteristics and, with membrane vesicles, membrane status, but these benefits were not reflected in improved fertility after cervical or intrauterine insemination.     

家养梅花鹿腹腔镜输精技术研究——CIDR+PMSG同期发情处理后输精时间是关键

2004年9月15日(第0天)给45只成年雌性东北梅花鹿阴道放置"阴道内孕酮释放装置"(CIDR),到第12天取出CIDR,同时肌肉注射孕马血清促性腺激素(PMSG),21只母鹿注射200 IU(1组),24只母鹿注射150 IU(2组)。CIDR取出后24~72 h,用带试情布的成年公鹿对处理母鹿进行持续试情。结果第1组和第2组同期发情率分别为86%(18/21)和92%(22/24)(P>0.05),取出CIDR至发情的平均时间分别为(47.54±9.33)h和(49.74±6.69)h(P>0.05)。第2组1只母鹿表现出孕期发情现象。对发情母鹿进行腹腔镜人工输精,输精时间为取出CIDR后的48~78 h。输精后第5天到11月15日,每天早晨、中午和傍晚进行试情,检查人工输精母鹿返情情况,结果第1组和第2组人工输精母鹿未返情率分别为89%(16/18)和76%(16/21)。两组母鹿产仔率分别为50%(9/18)和38%(8/21)(P>0.05)。这对同期发情梅花鹿的定时输精特别有意义。     

A study on the effect of GnRH administration on the ovarian response and laparoscopic intrauterine insemination of Awassi ewes treated with eCG to induce superovulation

The effect of GnRH administration on superovulatory response of ewes treated with equine chorionic gonadotrophin (eCG) in breeding and nonbreeding seasons and the contribution of laparoscopic insemination to the improvement of fertilization and embryo recovery were investigated. Twenty-four nonpregnant Awassi ewes of 3–4 years of age were randomly allocated into two groups (n = 12). Each ewe was treated with a progesterone impregnated intravaginal sponge for 12 days. The following superovulation treatment was used: ewes of group 1 received 1,200 IU of eCG once as an intramuscular injection 48 h prior to sponge withdrawal; ewes of group 2 also received 1,200 IU of eCG once as an intramuscular injection, 48 h prior to sponge withdrawal and after 24 h of sponge removal. Ewes were injected with 80 μg of GnRH. Ewes of groups 1 and 2 were further subdivided into four equal groups (n = 6). Subgroups A and C (superovulated with eCG and eCG plus GnRH, respectively) were mated naturally at least two times with Awassi rams of proven fertility at 8-h intervals. Subgroups B and D (same as A and C) had intrauterine insemination at 44–46 h after sponge removal, under laparoscopic visualization of uterine horns, depositing 1 ml of diluted semen containing 100 × 10⁶ motile sperm in the distal portion of each uterine horn. Ovarian response was assessed by determining the number of corpora lutea by laparoscopy at day 6 after mating. Embryo recovery was performed by using a semi-laparoscopic flushing procedure in both uterine horns. Results of the present study showed that ewes treated in breeding season with eCG plus GnRH has a higher number (P < 0.05) of corpora lutea than eCG alone as 7.33 ± 0.54 and 4.33 ± 0.39, respectively. There was no significant difference in the number of corpora lutea in nonbreeding season when ewes treated with eCG and eCG plus GnRH. The number of unovulated follicles was significantly higher (P < 0.05) in eCG treated ewes than in ewes treated with eCG plus GnRH, both in the breeding and nonbreeding seasons. The number of recovered embryos from ewes treated with eCG plus GnRH and eCG differ significantly (P < 0.05) as 4.32 ± 0.56 and 1.06 ± 0.26, respectively, in the breeding seasons. No significant difference was observed when these hormones used for superovulation in the nonbreeding season. A higher number of unfertilized ova (P < 0.05) was observed in ewes when naturally inseminated than in ewes inseminated using the intrauterine laparoscopic technique. Higher rate of embryo recovery (P < 0.05) was achieved when ewes were inseminated via intrauterine (4.66 ± 0.66) compared with ewes naturally mated (2.16 ± 0.74). The fertilization rate in ewes inseminated intrauterine using laparoscopic techniques and naturally mated were 91.5% and 44.8%, respectively. Fertilization failure in ewes inseminated intrauterine using laparoscopic techniques and naturally mated were 8.4% and 55.2%, respectively. It could be concluded that administration of GnRH 24 h after sponge removal increased ovulation rate of Awassi ewes treated with eCG for superovulation in the breeding season. The use of eCG to induce superovulation in Awassi ewes combined with laparoscopic intrauterine insemination increases the fertilization rate.     

Influence of a controlled internal drug release after fixed-time artificial insemination on pregnancy rates and returns to estrus of nonpregnant cows

We determined whether an ovulatory estrus could be resynchronized in previously synchronized, AI nonpregnant cows without compromising pregnancy from the previous synchronized ovulation or to those inseminated at the resynchronized estrus. Ovulation was synchronized in 937 suckled beef cows at 6 locations using a CO-Synch + progesterone insert (controlled internal drug release; CIDR) protocol [a 100-microg injection of GnRH at the time of progesterone insert, followed in 7 d by a 25-mg injection of PGF(2alpha) at insert removal; at 60 h after PGF(2alpha), cows received a fixed-time AI (TAI) plus a second injection of GnRH]. After initial TAI, the cows were assigned randomly to 1 of 4 treatments: 1) untreated (control; n = 237); 2) progesterone insert at 5 d after TAI and removed 14 d after TAI (CIDR5-14; n = 234); 3) progesterone insert placed at 14 d after TAI and removed 21 d after TAI (CIDR14-21; n = 232); or 4) progesterone insert at 5 d after TAI and removed 14 d after TAI and then a new CIDR inserted at 14 d and removed 21 d after TAI (CIDR5-21; n = 234). After TAI, cows were observed twice daily until 25 d after TAI for estrus and inseminated according to the AM-PM rule. Pregnancy was determined at 30 and 60 d after TAI to determine conception to the first and second AI. Pregnancy rates to TAI were similar for control (55%), CIDR5-14 (53%), CIDR14-21 (48%), and CIDR5-21 (53%). A greater (P < 0.05) proportion of nonpregnant cows was detected in estrus in the CIDR5-21 (76/110, 69%) and CIDR14-21 (77/120, 64%) treatments than in controls (44/106, 42%) and CIDR5-14 (39/109, 36%) cows. Although overall pregnancy rates after second AI service were similar, combined conception rates of treatments without a CIDR from d 14 to 21 [68.7% (57/83); control and CIDR5-14 treatments] were greater (P = 0.03) than those with a CIDR during that same interval [53.5% (82/153); CIDR5-21 and CIDR14-21 treatments]. We conclude that placement of a progesterone insert 5 d after a TAI did not compromise or enhance pregnancy rates to TAI; however, conception rates of nonpregnant cows inseminated after a detected estrus were compromised when resynchronized with a CIDR from d 5 or 14 until 21 d after TAI.     

Achieving canine pregnancy by using frozen or chilled extended semen

Successful artificial insemination in the dog requires good timing of the insemination, skilled collection and handling of the semen, and mastering of insemination techniques. The bitch should be inseminated late in estrus. The insemination dose should contain at least 150 to 200 x 10(6) spermatozoa. Fresh semen can be inseminated vaginally, whereas frozen-thawed semen should be inseminated into the uterus. Pregnancy rates of 84% with fresh semen and 69% with frozen semen are reported.     

Artificial insemination outcomes in beef females using bovine sperm with a detectable fertility-associated antigen

In this study, semen samples from 25 bulls that had passed a breeding soundness evaluation were analyzed for the presence or absence of a 31-kDa protein, known as fertility-associated antigen (FAA), on spermatozoal membranes. Eighteen bulls had FAA on sperm (FAA-positive) and seven were devoid of FAA on sperm (FAA-negative). A single ejaculate from each bull was extended and frozen with 25 to 30 x 10(6) sperm in .5-mL straws. Crossbred replacement heifers (n = 865) were estrus-synchronized and artificially inseminated either at timed AI or 12 h after they were detected in estrus. Mature cows (n = 285) were inseminated 12 h after they were detected in estrus during a 45-d AI period. Pregnancy rates (pooled) to first AI service for females (n = 764) inseminated with FAA-positive sperm were 65.6% and were 49.7% for females (n = 386) inseminated with FAA-negative sperm (P < .005). Among the estrus-synchronized replacement heifers, pregnancy rates to synchronized AI service for heifers (n = 550) inseminated with FAA-positive sperm were 62% and were 45.7% for heifers (n = 315) inseminated with FAA-negative sperm (P < .005). These data indicate that pregnancy rates to first AI service at spontaneous and synchronized estrus are higher when using semen from bulls with detectable FAA on spermatozoal membranes compared to semen from bulls devoid of FAA on membranes. Fertility-associated antigen is an important determinant for fertility potential of sperm from bulls to be used in AI breeding programs.     

Follicle culture enhances fertilizability and cleavage of bovine oocytes matured in vitro

The fertilization and cleavage of bovine oocytes matured by intra- or extra-follicular methods were investigated. Oocytes were fertilized in vitro or in the rabbit oviduct and cleavage was assessed after in vitro culture of in vitro fertilized oocytes and after in vivo culture (rabbit oviducts) of xenogenously fertilized oocytes. The effect of fertilization with fresh-diluted or frozen-thawed semen were also examined. The intra-follicular method did not increase the nuclear maturation rate as compared with the extra-follicular method (57.9 and 52.7%, respectively). However, the proportions of in vitro fertilized eggs (54.8%) and of cleaved eggs (two to eight cells; 34.6%) in the rabbit oviduct for 48 h after xenogenous fertilization were higher (P less than .025) in the intra-follicular oocytes than those of the extra-follicular oocytes (37.1 and 21.3%, respectively). It was also found that the use of fresh-diluted semen resulted in more cleaved eggs from the rabbit oviduct than the use of frozen-thawed semen (43.4 and 23.3% in the intra-follicular oocytes, P less than .025; 31.0 and 7.8% in the extra-follicular oocytes, P less than .05), while the appearance of cleaved eggs following in vitro fertilization was extremely low (0 to 6.6%). The present results demonstrated that the intra-follicular culture method of bovine oocytes provided a physiological environment for cytoplasmic maturation leading to higher fertilizability and development than the conventional in vitro culture of extra-follicular oocytes.     

Improvement by ergonovine of sperm transport, fertilization and pregnancy rates in ewes in natural or prostaglandin-induced estrus

Eight experiments were conducted with 451 ewes to test effects of ergonovine, prostaglandin F2 alpha (PGF2 alpha) and phenylephrine on sperm transport and fertility. In most experiments, ewes were mated at estrus and necropsied 2 or 3 h later. Sperm were flushed from the oviducts, uterus and anterior, middle and posterior thirds of the cervix and counted. Various doses of PGF2 alpha or phenylephrine given im at mating caused no significant increase in sperm numbers in any segment of the tract 2 h later. Three different dose levels of ergonovine were given im to ewes in natural estrus 1 h after mating and ewes were necropsied 3 h after mating. Doses of .2 and 1.0 mg were ineffective, but .5 mg increased sperm numbers about 10-fold in the oviducts and uterus. When given im at the time of artificial insemination, .6 mg of ergonovine increased the fertilization rate at 3 d from 5/25 in control ewes to 12/25 (P less than .05). In three experiments with ewes in PGF2 alpha-induced estrus, .6 mg of ergonovine increased sperm numbers in the cervix and uterus at 3 h after mating and in the uterus and oviducts at 23 h, near ovulation. Other ewes were artificially inseminated in the external cervical os and one-half of the ewes were given .6 mg of ergonovine im; ewes not returning to estrus were laparotomized at 22 to 26 d and embryos removed. After insemination during natural estrus with .2 ml of semen, pregnancy rates were 14/25 for control ewes and 15/25 for ergonovine-treated ewes; after insemination during natural estrus with .1 ml of semen, 6/35 and 18/35 (P less than .005); after insemination during PGF2 alpha-induced estrus with .2 ml of semen, 7/60 and 12/60. Fertilization and pregnancy rates combined were 32/145 (22%) for all control ewes and 57/145 (39%) for ergonovine-treated ewes (P less than .005).     

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.     

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.     

奶牛性控冻精人工授精影响因素研究

用分离X和Y精子的性控精液进行人工授精是控制家畜性别之最简单可行的方法.然而,低密度性控精液输精效果还不如常规人工授精,许多技术环节都有待改进.以常规冻精和稀释常规冻精为对照,研究解冻方法、输精时间和部位、不同精液来源和输精员以及育成和经产牛等因素对性控冻精人工授精妊娠率的影响.结果显示,精液解冻水浴温度和持续时间对人工授精效果有显著影响,性控精液对解冻水浴温度更敏感;性控冻精和稀释常规冻精比常规冻精对输精时间要求更严格;3种精液输精到排卵卵泡同侧子宫角基部受胎率都显著高于输精于子宫体和同侧子宫角前端;3种精液育成牛受胎率(80%)都显著高于经产牛(50%);于输精同时注射促排卵素3号明显提高性控冻精受胎率;经严格挑选、能够从事胚胎移植操作的技术熟练输精员之间性控冻精受胎率差异不显著;在所设计的不同条件下,性控冻精与稀释同样倍数的常规冻精行为相似,说明精子分离过程没有对精子造成特殊损伤.研究结果说明,精确控制人工授精各个技术环节可以实现消除性控与非性控、低密度与高密度精演之间的差别,获得高妊娠率.     

Endoscopic transcervical intrauterine artificial insemination in Labrador Retriever bitches

Besides post-thawing reduced semen quality, there are some difficulties in the execution of the endoscopic transcervical intrauterine artificial insemination (AI) with frozen-thawed semen in bitches. Therefore, the aims of the present study were to evaluate behavioral and reproductive parameters (i.e., vaginal cytology and serum progesterone level) to determine time of insemination and to investigate the particularities and difficulties of this technique in bitches using fresh semen. Ten Labrador Retriever bitches were submitted to three endoscopic transcervical intrauterine AIs (with 48 h intervals). Oestrus and ovulation period were established by behaviour evaluation, progesterone assays and vaginal cytology, enabling optimal timing for AI during oestrus. During AI, the following aspects were evaluated: cervical os catheterization difficulty, semen deposition resistance, occurrence of semen backflow, and time required to perform the AI. In this study, it was possible to catheterize the cervical os in all bitches, with different degrees of difficulty, by manipulating the equipment to allow cervical visualization and catheter introduction in the cervical canal. Serial serum progesterone assays enabled estimation of LH surge day, and thus of ovulation. The pregnancy rate was 90%, with a litter size of 5.0 ± 2.6 puppies per bitch. It was concluded that the difficulties in the execution of the endoscopic transcervical intrauterine AI technique in Labrador Retriever bitches were minimized by the equipment manipulation and practical experience.     

Laparoscopic intra-uterine insemination of fallow deer with frozen-thawed or fresh semen after synchronisation with CIDR devices

This study investigated the efficacy of fixed-time laparoscopic intra-uterine insemination of farmed fallow deer (Dama dama) with frozen-thawed or fresh semen. In the trials with frozen-thawed semen, a total of 547 mature non-lactating does across five New Zealand farms were used. For oestrous synchronisation and artificial insemination, a standard control regimen was applied to at least 30% of the does on each farm, involving the insertion of single CIDR type-G devices intravaginally for 14 days, deposition of 50 x 10(6) frozen-thawed spermatozoa at 65 hours after withdrawal of the CIDR device and the continuous presence of vasectomised bucks from the insertion of the CIDR device until 10 days after insemination. Various aspects of this protocol were changed for the remaining does on each farm, including inseminations at 60 or 70 hours, the absence of vasectomised bucks, insemination with 25 x 10(6) or 10 x 10(6) spermatozoa, synchronisation with CIDR type-S devices and synchronisation with prostaglandin. The conception rate, based on rectal ultrasonography at 45 days after insemination, was 67% across all treatments (n=547). Corrected conception rates (+/-s.e.), calculated following between-farm adjustments, were 67+/- 3% for the control regimen, 67+/- 9% and 73 +/- 8% for inseminations at 60 and 70 hours respectively, 61 +/- 9% for absence of bucks, 80 +/- 8% and 74 +/- 9% for inseminations with 25 x 10(6) and 10 x 10(6) spermatozoa respectively, 62 +/- 10% for CIDR type-S device synchronisation, and 49 +/- 10% for prostaglandin synchronisation. Despite apparent differences, none of the treatments resulted in adjusted conception rates that were significantly different from the control regimen (P>0.01). In the trials with fresh semen, 216 does in the USA were inseminated at 69-71 hours after withdrawal of the CIDR device using either cryopreserved semen from New Zealand (n=158; 25 x 10(6) spermatozoa per inseminate) or fresh semen (n=58; 7.5 x10(6) to 20 x 10(6) spermatozoa per inseminate) collected less than 10 hours earlier. The overall conception rates were 77% and 81% respectively, with no significant differences between semen type (frozen v. fresh) or fresh spermatozoa number per inseminate (P>0.01). A further 102 does in New Zealand similarly received fresh semen from 3/4 Mesopotamian buck. Doses of 10 x 10(6) (n=35), 5 x 10(6) (n=32) or 2.5 x 10(6) (n=35) spermatozoa per inseminate were delivered at 69-71 hours after withdrawal of the CIDR device. The conception rates were 77%, 66% and 51% respectively, reflecting a dose effect (P<0.05). However, 1/4 Mesopotamian does in the group (n=19) exhibited higher conception rates (95% overall) irrespective of semen dose, possibly indicating a semen/recipient genotype interaction. It is concluded that laparoscopic intra-uterine insemination of fallow deer with frozen-thawed or fresh semen at fixed intervals after removal of a CIDR device can give acceptable conception rates under a range of on-farm management options and semen doses.     

Effect of green buffer storage on the fertility of fresh camel semen after artificial insemination

Artificial insemination (AI) is one of the most widely used reproductive technologies, and there is considerably interest in commercializing this technology in camels. Storage of semen extender frozen (at -20 °C) is of considerable interest to scientists working with camels, as transportation of diluents at refrigeration temperature is not always possible given the hot, arid and remote conditions that dromedary camels exist in. Therefore, this study was conducted to compare the fertility of fresh camel semen, after dilution in fresh or frozen-thawed green buffer (GB), after AI into single and multiple ovulating female camels. No differences were observed in any sperm characteristics (motility, membrane integrity, acrosome integrity or morphology) when semen was diluted in fresh or frozen-thawed GB (p>0.05). Sperm motility was increased by dilution (fresh: 70.7 ± 4.9% and frozen: 68.8 ± 3.1%) compared with the motility of sperm in neat semen (35 ± 2.85%; p<0.05), and sperm motility changed from oscillatory to forward progressive after dilution. Pregnancy rates were higher (p<0.05) for single ovulating camels inseminated with semen diluted in fresh (72.7%) compared with frozen-thawed GB (27.3%), and fertilization rates were also higher (p<0.05) for multiple ovulating camels inseminated with semen diluted in fresh (83.3%) compared with frozen-thawed GB (11.1%). These results clearly demonstrate the detrimental effect of freezing and thawing semen diluent on the fertility of fresh camel semen. However, further studies are required to elucidate the mechanism responsible for this reduction in fertility. Moreover, these results demonstrate that the fertility of fresh camel semen diluted in fresh GB is high enough to be considered commercially viable.     

First Established Pregnancies in Mediterranean Italian Buffaloes (Bubalus bubalis) Following Deposition of Sexed Spermatozoa near the Utero Tubal Junction

At the time of AI following Ovsynch protocol, a total of 51 buffaloes were randomly divided in a first group (n = 30) subjected to conventional AI into the uterine body with 20 million non-sex sorted frozen-thawed spermatozoa, while a second group (n = 21) was inseminated near the utero-tubal junction (UTJ) ipsilateral to the ovary carrying the preovulatory follicle with 2.5 million live (4 million total) sex-sorted frozen-thawed spermatozoa. The semen used for flowcytometric sorting was collected and processed on a farm in Italy, and then shipped to a laboratory in Germany. Eleven buffaloes were inseminated with X-chromosome bearing spermatozoa and 10 with Y-chromosome bearing spermatozoa. Conception rates after conventional and UTJ inseminations were 43.3% (n = 13) and 42.8% (n = 9) respectively (p = 0.97). Eight of the nine foetuses obtained after insemination with sexed spermatozoa corresponded to the sex as predicted by the cell sorting procedure (five male and four female foetuses by ultrasound vs six male and three female foetuses by cell sorting). In conclusion, for the first time buffalo semen has been successfully subjected to procedures for flowcytometric sperm sorting and freezing. Low doses of sexed spermatozoa have been deposited near the UTJ giving conception rates similar to those of conventional AI with full dose.