Alternatives to improve a prostaglandin-based protocol for timed artificial insemination in sheep

The objective was to improve the reproductive performance of a prostaglandin (PG) F_2α-based protocol for timed artificial insemination (TAI) in sheep (Synchrovine®: two doses of 160 μg of delprostenate 7 d apart, with TAI 42 h after second dose). Three experiments were performed: Experiment 1) two doses of a PGF_2α analogue (delprostenate 80 or 160 μg) given 7 d apart; Experiment 2) two PGF_2α treatment intervals (7 or 8 d apart) and two times of TAI (42 or 48 h); and Experiment 3) insemination 12 h after estrus detection or TAI with concurrent GnRH. Experiments involved 1131 ewes that received cervical insemination with fresh semen during the breeding season (32/34 °S–58 °W). Estrous behaviour, conception rate, prolificacy, and fecundity (ultrasonography 30–40 d), were assessed. In Experiment 1, ewes showing estrus between 25 and 48 h or at 72 h after the second PGF_2α did not differ between 80 and 160 μg of delprostenate (73 vs 86%, P = 0.07; and 92 vs 95%, P = NS, respectively). Conception rate and fecundity were lower (P < 0.05) using 80 vs 160 μg (0.24 vs 0.42, and 0.27 vs 0.47, respectively). In Experiment 2, giving PGF_2α 7 d apart resulted in higher (P < 0.05) rates of conception (0.45 and 0.51) and fecundity (0.49 and 0.53) than treatments 8 d apart (conception: 0.33 and 0.29; fecundity: 0.33 and 0.34) for TAI at 42 and 48 h, respectively. In Experiment 3, rates of conception, prolificacy and fecundity were similar (NS) between Synchrovine® with TAI at 42 h (0.50, 1.13, and 0.56) and AI 12 h after estrus detection (0.47, 1.18, and 0.55), and Synchrovine® plus GnRH at TAI (0.38, 1.28, and 0.49). However, all TAI treatments had lower (P < 0.05) prolificacy and fecundity compared to AI following detection of spontaneous estrus (1.39 and 0.83, respectively). In conclusion, the Synchrovine® protocol was: a) more successful using 160 vs 80 μg delprostenate; b) more successful with a 7 d than 8 d PGF_2α interval; c) similarly effective for TAI versus AI 12 h after estrus detection; and d) not improved by giving GnRH at TAI.     

Insemination time and dilution rate of cooled and chilled ram semen affects fertility

Adult Merino ewes (n=448) were apportioned into two groups and inseminated with: extended at 30 degrees C with skim milk and stored for 6h at 15 degrees C (cooled semen) or extended with skim milk-citrate trisodium with egg yolk and stored for 24h at 5 degrees C (chilled semen). Each group was further subdivided according to the time of cervical insemination at 42, 46 and 50h after pessary (MAP-60 mg) removal and according to the dilution of the semen (120 x 10(6) spermatozoa in 0.05, 0.1 and 0.2 ml). The pregnancy rate after insemination with cooled semen was 50% better than that after chilled semen (56.7 vs. 37.5%; P<0.001). Pregnancy rate was not affected by the volume of insemination; however, there was a tendency of increased lambing rate with an insemination dose of 0.1 cc (1:2, dilution), especially when the ewes were inseminated with cooled semen. The effect of time on insemination was significant only in ewes inseminated with chilled semen at 5 degrees C (P<0.01). Insemination carried out 46 h after pessary removal resulted in higher pregnancy and lambing rate (36.5, 31.1; 52.0, 45.3; and 24.0, 20.0 at 42, 46 and 50h, respectively). Pregnancy of ewes inseminated with chilled semen at 46 h after pessary removal was similar to that obtained using cooled semen (52.0 vs. 56.7%). From this study, it is concluded that advancing the time of insemination with chilled semen at 5 degrees C improves pregnancy and that the lambing obtained under these conditions is similar to the one obtained with cooled semen.     

Fertility of ewes following artificial insemination with semen frozen in pellets or straws, a preliminary report

In two trials involving the artificial insemination of 194 ewes, the fertility of ram semen was examined following freezing, either in pellet form or in straws, and after storage in a chilled state (15 degrees C) for up to 16 hours. Estrus was synchronized in ewes by intravaginal sponge (MAP) treatment for 14 days. At sponge removal 600 IU PMSG was injected and the ewes received two inseminations 50 and 60 hours later. Fertility was assessed at lambing. In trial 1, the mean lambing rate of 52% (16 31 ) for semen frozen in pellets was higher than 29% (9 31 ) for semen frozen in straws but this difference was not significant. In trial 2, ewes inseminated with chilled semen and semen frozen in pellets had lambing rates of 83% (44 53 ) and 55% (44 79 ) respectively (P<0.001).     

Evaluation of two progestogen-based estrous synchronization protocols in yearling heifers of Bos indicus × Bos taurus breeding

Yearling Bos indicus × Bos taurus heifers (n = 410) from three locations, were synchronized with either the Select Synch/CIDR+timed-AI (SSC+TAI) or 7-11+timed-AI (7-11+TAI) treatments. On Day 0 of the experiment, within each location, heifers were equally distributed to treatments by reproductive tract score (RTS; Scale 1-5: 1 = immature, 5 = estrous cycling) and body condition score. The 7-11+TAI treatment consisted of melengestrol acetate (0.5 mg/head/d) from Days 0 to 7, with PGF_2α (25 mg im) on Day 7, GnRH (100 μg im) on Day 11, and PGF_2α (25 mg im) on Day 18. The SSC+TAI heifers received the same carrier supplement (without MGA) from Days 0 to 7, and on Day 11 they were given 100 μg GnRH and an intravaginal CIDR (containing 1.38 g progesterone). The CIDR were removed on Day 18, concurrent with 25 mg PGF_2α im For both treatments, estrus was visually detected for 1 h twice daily (0700 and 1600 h) for 72 h after PGF_2α, with AI done 6 to 12 h after a detected estrus. Non-responders were timed-AI and received GnRH (100 μg im) 72 to 76 h post PGF_2α. The 7-11+TAI heifers had a greater (P < 0.05) estrous response (55.2 vs 41.9%), conception rate (47.0 vs 31.3%), and synchronized pregnancy rate (33.5 vs 24.8%) compared to SSC+TAI heifers, respectively. Heifers exhibiting estrus at 60 h (61.7%) had a greater (P < 0.05) conception rate compared to heifers that exhibited estrus at ≤ 36 (35.3%), 48 (31.6%), and 72 h (36.2%), which were similar (P > 0.05) to each other. As RTS increased from ≤ 2 to ≥ 3, estrous response, conception rate, synchronized pregnancy rate, and 30 d pregnancy rate all increased (P < 0.05), irrespective of synchronization treatment. In conclusion, the 7-11+TAI treatment yielded greater synchronized pregnancy rates compared to SSC+TAI treatment in yearling Bos indicus × Bos taurus heifers.     

Artificial insemination of sheep: the fertility of semen extended in diluents containing egg yolk and inseminated soon after dilution or stored at 5 degrees C for 24 or 48 hours.

In an experiment involving the artificial insemination (AI) of 1175 ewes, ram semen was diluted 10- or 30-fold in a buffered glucosesaline solution containing either 1.5% or 6% (v/v) egg yolk. Part of each semen collection was used undiluted for control AI of 10⁸ sperm/dose. Diluted samples were reconcentrated to 10⁹ sperm/ml by centrifugation and, from these preparations, 10⁸ spermatozoa were inseminated in a standard volume of 100 μl. Fertility was assessed by 28–45 day non-returns to oestrus.The processes of dilution and reconcentration caused a significant drop in the non-return rate (NRR) and cooling to 5°C and storage for up to 48 hrs at this temperature gave a further large, and highly significant, reduction in NRR. There was no significant effect of level of egg yolk in the diluent on NRR.     

Fertility-associated antigen on Nelore bull sperm and reproductive outcomes following first-service fixed-time AI of Nelore cows and heifers

The objective was to determine whether the presence of fertility-associated antigen (FAA) on sperm collected from Nelore (Bos indicus) bulls can be used to assess potential fertility of sperm for use at first-service fixed-time AI (TAI). Six Nelore bulls were selected based on FAA status (FAA-negative: N = 3; FAA-positive: N = 3) and the ability to produce neat semen with ≥ 70% morphologically normal sperm and 60% estimated progressive motility before cryopreservation. In Experiment 1, suckled multiparous Nelore cows (N = 835) were evaluated for body condition score (BCS) and received an intravaginal progesterone device (CIDR) and 2.0 mg of estradiol benzoate (Day 0). On Day 9 the CIDR was removed, 12.5 mg of PGF_2α and 0.5 mg of estradiol cypionate were administered, and calves were removed for 48 h. All cows received TAI on Day 11 (48 h after CIDR removal). Pregnancy per TAI (P/TAI) was not different between FAA-positive and FAA-negative bulls (41.5% vs. 39.3%, respectively). There was an effect of AI technician on P/TAI (36.0% vs. 43.9%; P < 0.05) and BCS tended to affect P/TAI (P = 0.09), as cows with BCS ≥ 2.75 were 1.4 times more likely to become pregnant compared with cows with BCS < 2.75. In Experiment 2, nulliparous Nelore heifers (N = 617) were evaluated for BCS and received a CIDR and estradiol benzoate (2.0 mg) on Day 0. On Day 7, all heifers received PGF_2α (12.5 mg). On Day 9, CIDR inserts were removed and all heifers received estradiol cypionate (0.6 mg) and 200 IU eCG. All heifers received TAI on Day 11 (48 h after CIDR removal). Pregnancy/TAI was different (P = 0.04) between FAA-positive and FAA-negative bulls (33.7% vs. 40.7%, respectively). Presence of FAA on sperm was unsuccessful in assessing the potential fertility of sperm for use in TAI.     

Fertility of swamp buffalo following the synchronization of ovulation by the sequential administration of GnRH and PGF2alpha combined with fixed-timed artificial insemination

This study evaluated fertility in swamp buffalo after synchronization of ovulation combined with fixed time artificial insemination. At the start of the study, designated day 0, from a group of 98 female Thai swamp buffalo, 55 buffalo (heifers n° = 20 and cows n° = 35) were selected to be synchronized with GnRH (Day 0) followed by PGF₂alpha (Day 7) and a second treatment with GnRH (Day 9). All buffalo were inseminated at two fixed times 12 h and 24 h after the second injection of GnRH (Ovsynch+TAI group); a second group of 43 buffalo (heifers n° = 19 and cows n° = 24) were not treated and were artificially inseminated (AI) at natural estrus (AI group). Blood samples were taken 22 days after insemination to evaluate progesterone plasma levels. In the Ovsynch+TAI group, overall conception rate (CR; i.e. the number of cows with progesterone >4.0 ng/ml on day 22 after AI divided by the number of animals inseminated), was 38.1% and overall pregnancy rate (PR; i.e. the number of cows that were pregnant at day 50-60 after insemination divided by the number of animals inseminated), was 32.7%. In the AI group overall CR and PR was 34.9%.Within the Ovsynch+TAI group, CR and PR were reduced (P < 0.05) in heifers compared with cows (CR 15.0% vs. 51.4% for heifers and cows, respectively; PR 15.0% vs. 42.9% for heifers and cows, respectively). Within the AI group the efficacy of treatment was similar between heifers and cows (CR and PR 31.6% for heifers and 37.5% for cows).In conclusion, this study indicates that in swamp buffalo it is possible to synchronize ovulation and use timed artificial insemination with the Ovsynch+TAI protocol.     

Incorporation of a rapid pregnancy-associated glycoprotein ELISA into a CIDR-Ovsynch resynchronization program for a 28 day re-insemination interval

The objective was to compare two resynchronization programs; one that used a blood-based ELISA for pregnancy-associated glycoproteins (PAG) for pregnancy diagnosis so that non-pregnant cows were re-inseminated at 28 d after first TAI, and another that used transrectal ultrasonography for pregnancy diagnosis so that non-pregnant cows were re-inseminated at 35 d after first TAI. The PAG_resynch cows (n = 103) began CIDR-Ovsynch resynchronization on Day 18 after first TAI (Day 0). On Day 25, the CIDR was removed and pregnancy diagnosis with a PAG ELISA was performed. If a cow was not pregnant on Day 25, she was treated with PGF_2α, treated with GnRH 2 d later (Day 27), and TAI on Day 28. Control cows (n = 99) were observed for estrus until Day 25, when they began an identical CIDR-Ovsynch program with pregnancy diagnosis by transrectal ultrasonography on Day 32. If a cow was not pregnant on Day 32, then she was treated with PGF_2α, treated with GnRH 2 d later (Day 34), and TAI on Day 35. There was no difference in pregnancy per AI (P/AI) for either group at first or second insemination. For cows without pregnancy loss, the interval between first and second (P < 0.001) or second and third (P < 0.016) TAI was shorter for PAG_resynch cows compared with Control cows. The interval between first and second or second and third TAI was not different if pregnancy loss cows were included in the analysis. Plasma progesterone concentrations were similar at PGF_2α treatment, and plasma estradiol concentrations increased similarly after PGF_2α treatment for PAG_resynch and Control cows. In conclusion, the 28 d CIDR-Ovsynch resynchronization protocol was comparable to a 35 d CIDR-Ovsynch resynchronization protocol that also included estrus detection. Shortened resynchronization protocols that do not require estrus detection may improve reproductive efficiency in dairy cattle.     

Artificial insemination of sheep: the effect of semen diluents containing egg yolk on the fertility of ram semen.

Results from an artificial insemination (AI) experiment revealing the effect of semen dilutents containing egg yolk on the fertility of ram semen are presented. Ram semen was diluted 30-fold in buffered glucose-saline solution containing . 375, 1.5, or 6% v/v egg yolk and a portion of each was used soon for the AI of ewes or was incubated at 35 degrees C for 1 hour prior to AI. Some of the semen collection was used undiluted for AI of 10(8) spermatozoa/dose. All diluted samples were reconcentrated by centrifugation so that each dose was 10(8) spermatozoa in a volume of 100 mcl. 1146 ewes were inseminated. Fertility was assessed from 28 to 45 day nonreturns to estrus and nonreturn rates (NRRs) were expressed as percentages for the various treatments. Undiluted semen (controls) revealed 69%, semen used soon after dilution, .375% yolk in dilutent 58%, 1.5% yolk 50%, 6% yolk 42%; diluted semen incubated for 1 hour before use, .375% yolk 49%, 1.5% yolk 51%, and 6% yolk 39%. NRR was significantly depressed by dilution (p less than .001) and by increasing amounts of egg yolk (p less than .01) in the dilutent. Incubation of diluted semen before AI caused a small fall in NRR.     

Strategies to improve pregnancy per insemination using sex-sorted semen in dairy heifers detected in estrus

The objective was to improve pregnancy per artificial insemination (P/AI; 35-42 d after AI) in virgin Jersey heifers bred by AI of sex-sorted semen after being detected in estrus. Giving 100 μg of GnRH at first detection of estrus, with AI 12 h later, did not affect P/AI in Experiment I [GnRH = 47.2% (100/212) vs. No GnRH = 51.7% (104/201); P = 0.38] or Experiment II [GnRH = 53.1% (137/258) vs. No GnRH = 48.6% (122/251); P = 0.43]. In these two experiments, estrus detection was done with tail-head chalk or a HeatWatch^® system, respectively. In Experiment III, a single insemination dose (2.1 × 10⁶ sperm) 12 h after estrus detection (n = 193), a double dose at 12 h (n = 193), or a double dose involving insemination 12 and 24 h after estrus detection (n = 190) did not affect P/AI (87/193 = 45.1%, 85/193 = 44.0%, and 94/190 = 49.5%, respectively; P = 0.51). However, P/AI was influenced by the number of AI service (First, 115/208 = 55.3%^a; Second, 94/204 = 46.1%^a; and Third, 57/165 = 34.8%^b; P = 0.004). In Experiment IV, the P/AI of heifers inseminated from 12 to 16 h after the onset of estrus (40/106 = 37.7%) was less (P = 0.03) than those inseminated from 16.1 to 20 h (85/164 = 51.8%), and 20.1 to 24 h (130/234 = 55.6%). However, the P/AI for heifers inseminated from 24.1 to 30 h (61/134 = 45.5%) did not differ from that of any other interval. In conclusion, in Jersey heifers inseminated with sex-sorted semen, P/AI was not significantly affected by giving GnRH at detection of estrus or a double insemination dose, but it was higher with AI 16.1 to 24 h vs. 12 to 16 h after the onset of estrus.     

Comparison of progesterone-based protocols with gonadotropin-releasing hormone or estradiol benzoate for timed artificial insemination or embryo transfer in lactating dairy cows

The objective was to compare two protocols for synchronizing ovulation in lactating Holstein cows submitted to timed AI (TAI) or timed ET (TET). Within each farm (n = 8), cows (n = 883; mean ± SEM 166.24 ± 3.27 d postpartum, yielding 36.8 ± 0.34 kg of milk/d) were randomly assigned to receive either: 1) an intravaginal progesterone insert (CIDR^®) with 1.9 g of progesterone + GnRH on Day -10, CIDR^® withdrawal + PGF2α on Day -3, and 1 mg estradiol cypionate on Day -2 (treatment GP-P-E; n_TAI = 180; n_TET = 260); or 2) a CIDR^® insert + 2 mg estradiol benzoate on Day -10, PGF2α on Day -3, CIDR^® withdrawal + 1 mg estradiol cypionate on Day -2 (treatment EP-P-E; n_TAI = 174; n_TET = 269). Cows were subsequently randomly assigned to receive either TAI on Day 0 or TET on Day 7. Serum progesterone concentration on Day -3 was greater in GP-P-E than in EP-P-E (2.89 ± 0.15 vs 2.29 ± 0.15 ng/mL; P < 0.01), with no significant effect of group on serum progesterone on Day 7. Compared to cows submitted to TAI, those submitted to TET had greater pregnancy rates on Day 28 (44.0% [233/529] vs 29.7% [105/354]; P < 0.001) and on Day 60 (37.6% [199/529] vs 26.5 [94/354]; P < 0.001). However, there were no effects of treatments (GP-P-E vs EP-P-E; P > 0.10) on synchronization (87.0% [383/440] vs 85.3% [378/443]), conception (TAI: 35.3% [55/156] vs 33.8% [50/148]; TET: 50.7% [115/227] vs 51.3% [118/230]) and pregnancy rates on Days 28 (TAI: 30.5% [55/180] vs 28.7% [50/174]; TET: 44.2% [115/260] vs 43.9% [118/269]) and 60 (TAI: 27.2% [49/80] vs 25.9% [45/174]; TET: 38.8% [101/260] vs 36.4% [98/269]). In conclusion, GP-P-E increased serum progesterone concentrations on Day -3, but rates of synchronization, conception, and pregnancy were not significantly different between cows submitted to GP-P-E and EP-P-E protocols, regardless of whether they were inseminated or received an embryo.     

Estrus synchronization and artificial insemination of hair sheep ewes in the tropics

Hair sheep ewes (St. Croix White and Barbados Blackbelly) were used to evaluate 3 methods of estrus synchronization for use with transcervical artificial insemination (TAI). To synchronize estrus, ewes (n = 18) were treated with PGF2alpha (15 mg, im) 10 d apart, with controlled internal drug release (CIDR) devices containing 300 mg progesterone for 12 d (n = 18), or with intravaginal sponges containing 500 mg progesterone for 12 d (n = 18). On the day of the second PGF2alpha injection or at CIDR or sponge removal, sterile rams were placed with the ewes. Jugular blood samples were collected from the ewes at 6-h intervals until the time of ovulation, and daily for 16 d after estrus (Day 0). Plasma was harvested and stored at -20 degrees C until LH, and progesterone concentrations were determined by RIA. There was no difference (P>0.10) in time to estrus among the CIDR-, PGF2alpha- or sponge-treated ewes. All of the ewes in the CIDR group and 94.4% of the sponge treated ewes exhibited estrus by 36 h after ram introduction, while only 72.2% of PGF2alpha-treated ewes showed signs of estrus by this time (P<0.06). The time from ram introduction to ovulation was not different (P>0.10) among the CIDR-, PGF2alpha- or sponge-treated ewes. The time to the preovulatory LH surge was similar (P>0.10) among CIDR, PGF2alpha and sponge treated ewes. Progesterone levels through Day 16 after the synchronized estrus were not different (P>0.10) among treatment groups. Hair sheep ewes (n = 23) were synchronized using PGF2alpha and bred by TAI using frozen-thawed semen 48 h after the second injection. The conception rate to TAI was 2/23 (8.7%) and produced 3 ram lambs. In a subsequent trial, 17 ewes were synchronized with CIDR devices and bred by TAI using frozen-thawed semen 48 h after CIDR removal, resulting in a conception rate of 52.9% (9/17). It is possible to synchronize estrus in hair sheep using either CIDRs, sponges or PGF2alpha. Even though there were no significant differences in the timing of ovulation or the LH surge among the treatment groups, a higher conception rate was achieved in ewes synchronized with CIDR devices during the second trial. This may reflect an increase in the skill level of the TAI technician.     

Factors affecting pregnancy rates following laparoscopic insemination of 28,447 Merino ewes under commercial conditions: a survey

The results of laparoscopic insemination of 28,447 Australian Merino ewes with semen from 468 rams were used to study factors influencing pregnancy. The overall pregnancy rate was 71.7% (20,423/28,447). Pregnancy rates varied with type of progestagen implant, type and dosage of PMSG, fresh or frozen semen, wool type and number of ewes inseminated per hour. The pregnancy rate (64.6%) obtained with Medroxy-progesterone acetate (MAP) sponges, was significantly (P < 0.01) lower than with Fluorogestone acetate 30 mg (FGA 30; 74.7%) sponges, Fluorogestone acetate 40 mg (FGA 40; 72.1%) sponges, and Controlled Internal Drug Release (CIDR-G; 71.7%) implants. A PMSG dose of 200 IU resulted in significantly (P < 0.05) lower pregnancy rates (62.4%) compared with 250 IU (72.9%), 300 IU (79.1%) and > or = 375 IU (69.4%). The mean pregnancy rate for ewes administered Folligon PMSG was 71.9%, which was significantly higher (P < 0.001) than that of ewes treated with Pregnecol PMSG (65.8%). The use of Pregnecol PMSG and MAP sponges was associated, and thus their conditional effects could not be calculated. Ewes inseminated with fresh semen were significantly (P < 0.001) more likely to become pregnant (82.2%) than those inseminated with semen frozen in pellets (69.5%) or straws (71.6%). Ewes inseminated during the months of March, April or May (fall, 71.5%) were just as likely to become pregnant as those ewes inseminated in November, December, January or February (69.6%). Significantly (P < 0.05) fewer strong wool ewes become pregnant to laparoscopic AI, (67.6%) than fine (71.7%), fine medium (73%) or medium wool ewes. Significantly (P < 0.0001) more pregnancies (77.6%) were achieved when more than 55 ewes were inseminated per hour compared with fewer than 35 ewes per hour (63.4%).     

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.     

Influence of centrifugation or low extension rates prefreezing on the fertility of ram semen after cervical insemination

We compared the fertility of thawed ram semen, frozen according to different prefreezing semen handling protocols and previously well-defined in vitro, after cervical artificial insemination (AI) during natural estrus in Corriedale sheep. Following primary extension 1 + 1, we adjusted the final sperm concentration before packaging (200 x 10(6)/straw) either by centrifugation, in order to reconcentrate the extended semen (Protocol 1: P1), or without centrifugation, by adjusting the final sperm number by stepwise extension (Protocol 2: P2). We evaluated sperm motility (assessed both subjectively and with a computer-assisted sperm analysis instrument [CASA]), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline [CTC]) in vitro in three pooled straws of frozen-thawed semen. Three hundred Corriedale ewes, having shown spontaneous estrus during the breeding season (i.e., April, in the southern hemisphere) under extensive management conditions in Uruguay, were cervically inseminated with thawed semen from the same freezing operations as studied in vitro. The semen evaluation in vitro yielded higher percentages (P < 0.05) of damaged spermatozoa in the samples where sperm numbers were adjusted by extension before freezing (P2), compared with when adjustment was done by centrifugation (P1). However, due to the higher sperm concentration finally achieved by P2, the calculated total number of viable spermatozoa was almost equal in the two AI doses. We observed no differences in fertility between P1 and P2 for either nonreturn rates (NRRs) 21 (30.8 vs. 29.7%) and 36 (28.5 vs. 27.8%) days after AI or lambing rate (21.9 vs. 21.4%), respectively. Fertility did not differ significantly between the two different procedures of adjusting sperm numbers prior to freezing. This may indicate that the simplified protocol with adjusted extension of the semen, resulting in higher numbers of viable spermatozoa, should be the procedure of choice when freezing ram semen under field conditions. Further studies aimed at improving the modified protocol need to be performed.     

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.     

Effect of increasing GnRH and PGF2α dose during Double-Ovsynch on ovulatory response,luteal regression,and fertility of lactating dairy cows

Ovsynch-type synchronization of ovulation protocols have suboptimal synchronization rates due to reduced ovulation to the first GnRH treatment and inadequate luteolysis to the prostaglandin F_2α (PGF_2α) treatment before timed artificial insemination (TAI). Our objective was to determine whether increasing the dose of the first GnRH or the PGF_2α treatment during the Breeding-Ovsynch portion of Double-Ovsynch could improve the rates of ovulation and luteolysis and therefore increase pregnancies per artificial insemination (P/AI). In experiment 1, cows were randomly assigned to a two-by-two factorial design to receive either a low (L) or high (H) doses of GnRH (Gonadorelin; 100 vs. 200 μg) and a PGF_2α analogue (cloprostenol; 500 vs. 750 μg) resulting in the following treatments: LL (n = 263), HL (n = 277), LH (n = 270), and HH (n = 274). Transrectal ultrasonography and serum progesterone (P4) were used to assess ovulation to GnRH1, GnRH2, and luteal regression after PGF_2α during Breeding-Ovsynch in a subgroup of cows (n = 651 at each evaluation). Pregnancy status was assessed 29, 39, and 74 days after TAI. In experiment 2, cows were randomly assigned to LL (n = 220) or HH (n = 226) treatment as described for experiment 1. For experiment 1, ovulation to GnRH1 was greater (P = 0.01) for cows receiving H versus L GnRH (66.6% [217/326] vs. 57.5% [187/325]) treatment, but only for cows with elevated P4 at GnRH1. Cows that ovulated to GnRH1 had increased (P < 0.001) fertility compared with cows that did not ovulate (52.2% vs. 38.5%); however, no effect of higher dose of GnRH on fertility was detected. The greater PGF_2α dose increased luteal regression primarily in multiparous cows (P = 0.03) and tended to increase fertility (P = 0.05) only at the pregnancy diagnosis 39 days after TAI. Overall, P/AI was 47.0% at 29 days and 39.7% at 74 days after TAI; P/AI did not differ (P = 0.10) among treatments at 74 days (LL, 34.6%; HL, 40.8%; LH, 42.2%; HH, 40.9%) and was greater (P < 0.001) for primiparous cows than for multiparous cows (46.1% vs. 33.8%). For experiment 2, P/AI did not differ (P = 0.21) between H versus L treatments (44.2% [100/226] vs. 40.5% [89/220]). Thus, despite an increase in ovulatory response to GnRH1 and luteal regression to PGF_2α, there were only marginal effects of increasing dose of GnRH or PGF_2α on fertility to TAI after Double-Ovsynch.     

Endogenous prostaglandin F2α concentrations in bovine whole semen, seminal plasma, and extended semen

A series of experiments were conducted to quantify PGF_2α in bovine semen, seminal plasma, and extended semen, and to determine if PGF_2α was synthesized or released during extension of bovine semen. Concentrations of PGF_2α were measured in paired samples of whole and extended semen from beef and dairy bulls. Concentrations of PGF_2α did not differ between beef and dairy (mean ± SEM, 273.8 ± 42.8 vs. 210.3 ± 18.5 pg/mL, respectively; P = 0.12), but tended (P = 0.08) to be greater for whole compared with extended semen (255.5 ± 29.8 vs. 194.5 ± 17.0 pg/mL). Whole semen was extended at eight dilution rates (regardless of initial sperm concentration), using a diluent consisting of two fractions. Samples collected after each dilution step resulted in four subsamples. Concentrations of PGF_2α in subsamples decreased (P < 0.001) at higher dilution rates and later steps of extension. Subsequently, whole semen and seminal plasma were extended at three dilution rates. Initial PGF_2α concentration was greater (P < 0.001) for whole semen compared with seminal plasma. During extension, PGF_2α synthesis or release resulted in less disparity, but the amount synthesized or released was greater (P = 0.03) for semen compared with seminal plasma. We concluded that synthesis or release of PGF_2α during extension resulted in concentrations similar to whole semen.     

Effects of flunixin meglumine, recombinant bovine somatotropin and/or human chorionic gonadotropin on pregnancy rates in Nelore cows

The objective was to compare pharmacological strategies aiming to inhibit prostaglandin F2 alpha (PGF_2α) synthesis (flunixin meglumine; FM), stimulate growth of the conceptus (recombinant bovine somatotropin; bST) and progesterone (P₄) synthesis (human chorionic gonadotropin; hCG), as well as their combinations, regarding their ability to improve pregnancy rates in beef cattle. Lactating Nelore cows (N = 975), 35 to 70 days postpartum, were synchronized and inseminated by timed artificial insemination (TAI) on Day 0. On Day 7, cattle were allocated into eight groups and received one of the following treatments: saline (S) on Days 7 and 16 (Group Control); S on Day 7 and FM on Day 16 (Group FM); bST on Day 7 and S on Day 16 (Group bST); bST on Day 7 and FM on Day 16 (Group bST + FM); hCG on Day 7 and S on Day 16 (Group hCG); hCG on Day 7 and FM on Day 16 (Group hCG + FM); bST and hCG on Day 7 and S on Day 16 (Group bST + hCG), or bST and hCG on Day 7 and FM on Day 16 (Group bST + hCG + FM). The aforementioned treatments were administered at the following doses: 2.2 mg/kg FM (Banamine^®; Intervet Schering-Plough, Cotia, SP, Brazil), 500 mg bST (Boostin^®; Intervet Schering-Plough), and 2500 IU hCG (Chorulon^®; Intervet Schering-Plough). Pregnancy diagnosis was performed 40 days after TAI by transrectal ultrasonography. Pregnancy rates were not significantly different among treatments. However, there was a main effect of hCG treatment to increase pregnancy rates (63.0 vs. 55.4%; P = 0.001). Concentrations of P₄ did not differ significantly among groups on Day 7 or on Day 16. However, consistent with the higher pregnancy rates, hCG increased P₄ concentrations on Day 16 (10.6 vs. 9.6 ng/mL, respectively; P = 0.05). We concluded that hCG treatment 7 days after TAI improved pregnancy rates of lactating Nelore cows, possibly via a mechanism leading to induction of higher P₄ concentrations, or by reducing the luteolytic stimulus during maternal recognition of pregnancy.     

Long-term motility and fertility conservation of chilled canine semen using egg yolk added Tris-glucose extender: in vitro and in vivo studies

The effects of medium exchange on motility parameters of chilled canine semen preserved in egg yolk Tris-glucose (EYTG) extender were analyzed over a 27-d period. Semen extender was exchanged at three time points (Days 11, 21 and 27) after collection, when motility parameters were demonstrated to significantly decrease from parameters observed at semen preparation (Day 0) or at day of previous extender exchange. In the absence of medium exchanges, motile spermatozoa were observed up to Day 16 (mean +/- S.D. 1.5 +/- 0.3% of motile spermatozoa). A stimulation of the different semen motility parameters was observed after extender exchange. Semen extender exchange at Day 11 allowed conservation of motility until Day 21, compared to 16 d in the absence of extender exchange. At Day 21, when spermatozoa appeared immobile or dead, a second extender exchange was performed, allowing the extension of motility conservation up to Day 27. The third extender exchange, performed at Day 27, was no longer associated with motility stimulation. Glucose content in the medium decreased slowly over time; a concomitant decrease in pH was also observed. No changes in osmolarity were observed over time. To verify the fertility of long-term conserved chilled semen, two groups of 10 bitches were inseminated either once (Group 1) or twice at 48-h intervals (Group 2) intra-vaginally with semen conserved chilled for a mean of 9 +/- 1.8 d. Out of the 10 bitches inseminated once, 5 became pregnant, versus 7 in the group of animals inseminated twice. The present study reports the possibility to extend the conservation of chilled canine semen up to 3 wk with conservation of good fertility for at least 10 d. The role of energetic substrate and pH alteration is postulated and the classically accepted relation of semen motility/viability is raised.