J. Gil

Effect of cooling rates on post-thaw sperm motility, membrane integrity, capacitation status and fertility of dairy bull semen used for artificial insemination in sweden

We studied the effects of 2 different cooling rates during equilibration of semen from room temperature to 4 degrees C, at 4.2 degrees C/min (control split sample) or at 0.1 degree C/min (treatment split sample) on in vitro sperm viability post thawing and fertility after AI. Forty batches of split-frozen semen from 14 dairy bulls (Swedish Red and White breed) aged 14 to 16 m.o. or 66 to 79 m.o. were evaluated post-thawing for sperm motility (visual and computer-assisted sperm analysis [CASA], membrane integrity (fluorescent microscopy and flow cytometry post-loading with the combined fluorophores Calcein AM/EthD-1 and SYBR-14/PI); acrosomal status (with Pisum sativum agglutinin [PSA] staining); and capacitation status (CTC-assay). Fertility values (56-d nonreturn rate) of the slow cooling batches (treatment) were 0.4% units higher than for faster cooled (control) batches, but the difference was not statistically significant. Fertility values for the older bulls were 1.6% units higher than for the group of younger sires. No statistically significant correlations were found between semen viability parameters assessed in vitro and 56-d nonreturn rate. Visually assessed sperm motility, membrane integrity, capacitation and acrosomal status post-thawing did not differ significantly between cooling procedures, however the percentage of motile spermatozoa and the kinetic characteristics of spermatozoa--average path velocity (VAP), straight path velocity (VSL) and curvilinear velocity (VCL)--assessed by CASA differed significantly between cooling procedures. The results indicate that most of the in vitro sperm viability parameters post-thawing and the fertility results for bulls after AI did not differ significantly between the 2 semen cooling procedures tested.

Influence of centrifugation and different extenders on post-thaw sperm quality of ram semen

Using a 2-step extension methodology to freeze ram semen, 2 freezing protocols (P1 and P2) and 3 extenders were evaluated in a split-sample experiment. The freezing protocols were tested in combination with Extenders A and B (Experiment 1), and B and C (Experiment 2). Protocol 1 included centrifugation before filling the straws to reconcentrate the diluted semen to a calculated sperm concentration of 800 x 10(6) cells/mL. Protocol 2 involved appropriate ejaculate extension to yield 800 x 10(6) cells/mL as in P1, albeit avoiding centrifugation. Extenders A and B were milk-based and were supplemented with 5% egg yolk and fructose. Extender B was clarified by centrifugation (twice at 3310 g/20 min). Extender C was based on TRIS-citrate-fructose supplemented with 20% egg yolk and clarified as described for Extender B. Final glycerol concentration was 7% for all 3 extenders. Post-thaw parameters studied were subjective motility, computer assisted sperm motility analysis (CASA), membrane integrity (SYBR-14/P1), and capacitation status (chlortetracycline assay, CTC). The overall sperm concentration (x 10(6)/straw) differed (P<0.001) between P1 (mean+/-SD, 138.1+/-14.8) and P2 (216.5+/-13.9). Despite centrifugation, P1 appeared to be less harmful for spermatozoa than P2, yielding higher percentages of subjective motility, linearity, membrane integrity and uncapacitated spermatozoa. Due to the difference in concentrations obtained between P1 and P2, the total calculated numbers of spermatozoa having desirable characteristics were higher in samples processed as P2. In Experiment 1, P1 resulted in lower calculated numbers x 10(6) in the Aldose of subjective motility (87.2+/-5.1 vs 125.3+/-5.1; P<0.05), linearity (70.6+/-4.3 vs 79.8+/-4.3; NS), intact-membrane (77.4+/-5 vs 108.5+/-5.1; P<0.001), and uncapacitated (36.5+/-2.5 vs 46.5+/-2.5; P<0.05) spermatozoa, than P2. In Experiment 2, calculated sperm numbers (x 10(6)/straw) were lower in P1 than in P2 for subjective motility (80.8+/-5.4 vs 92.0+/-5.4; NS), linearity (63.3+/-5.6 vs 73.1+/-5.6; NS), membrane integrity (77.7+/-3.6 vs 101.0+/-3.6; P<0.001), and uncapacitated spermatozoa (28.3+/-3.24 vs. 4.1+/-3.2; P<0.01). Extender B (clarified milk extender) was consistently better than Extender A (nonclarified milk extender) for all parameters studied, but the difference was only statistically significant for linearity after 1 h of incubation at 38 degrees C (44.0+/-2.4 vs 36.2+/-2.4; P<0.05). Extender B was also better than Extender C (TRIS-citrate-fructose) for percentage of uncapacitated (49.7+/-2.2 vs 34.4+/-2.3; P<0.001), subjective motile (57.5+/-2.7 vs 43.8+/-2.7; P<0.01), and linear motile (46.5+/-2.8 vs 33.7+/-2.8; P<0.01) spermatozoa, but not for membrane integrity (51.6+/-1.5 vs 51.7+/-1.5). It was concluded that exclusion of centrifugation, as in P2, yielded higher sperm numbers with desirable characteristics per straw. Clarification of milk-based extender (B) resulted in better post-thaw sperm quality, especially compared with TRIS-based extender (C).

The use of prostaglandins in controlling estrous cycle of the ewe: A review.

This review considers the use of prostaglandin F(2α) and its synthetic analogues (PG) for controlling the estrous cycle of the ewe. Aspects such as phase of the estrus cycle, PG analogues, PG doses, ovarian follicle development pattern, CL formation, progesterone synthesis, ovulation rate, sperm transport, embryo quality, and fertility rates after PG administration are reviewed. Furthermore, protocols for estrus synchronization and their success in timed AI programs are discussed. Based on available information, the ovine CL is refractory to PG treatment for up to 2 days after ovulation. All PG analogues are effective when an appropriate dose is given; in that regard, there is a positive association between the dose administered and the proportion of ewes detected in estrus. Follicular response after PG is dependent on the phase of the estrous cycle at treatment. Altered sperm transport and low pregnancy rates are generally reported. However, reports on alteration of the steroidogenic capacity of preovulatory follicles, ovulation rate, embryo quality, recovery rates, and prolificacy, are controversial. Although various PG-based protocols can be used for estrus synchronization, a second PG injection improves estrus response when the stage of the estrous cycle at the first injection is unknown. The estrus cycle after PG administration has a normal length. Prostaglandin-based protocols for timed AI achieved poor reproductive outcomes, but increasing the interval between PG injections might increase pregnancy rates. Attempts to improve reproductive outcomes have been directed to provide a synchronized LH surge: use of different routes of AI (cervical or intrauterine), different PG doses, and increased intervals between PG injections. Finally we present our point of view regarding future perspectives on the use of PG in programs of controlled sheep reproduction.

Comparison of prostaglandin- and progesterone-based protocols for timed artificial insemination in sheep.

The objective was to compare the reproductive performance of a new PGF(2α)-based timed artificial insemination (TAI) protocol in sheep (Synchrovine®: two doses of PGF(2α), 7 d apart) to a traditional progesterone-eCG (P4-eCG) protocol, considering the effects of seminal state, AI-times, and AI-pathway. Three experiments involving 1297 multiparous Australian Merino ewes were done during the physiologic breeding season (location 32 °S-57 °W). Reproductive performance was assessed as non-return rate to service 21 d after AI (NRR21d), based on detection with androgenized wethers, as well as Fertility (pregnant/inseminated ewes), Prolificacy (fetuses/pregnant ewe), and Fecundity (fetuses/inseminated ewe), which were based on transabdominal ultrasonography 50 d after TAI. In Experiment 1, Synchrovine® treated ewes TAI cervically with fresh semen at 42, 48, or 54 h had similar NRR21d (0.51, 0.46, 0.57), Fertility (0.27, 0.31, 0.26), and Fecundity (0.29, 0.32, 0.27), all of which were lower (P < 0.05) than in a control P4-eCG group inseminated at 54 h (0.61, 0.48, 0.52, NRR21d, Fertility and Fecundity respectively). In Experiment 2, using chilled semen and cervical TAI, Synchrovine® treated ewes inseminated at 42 h yielded lower (P < 0.05) NRR21d, Fertility and Fecundity (0.28, 0.06, 0.06) compared to 48 (0.43, 0.24, 0.24) and 54 h (0.44, 0.22, 0.23). In Experiment 3 with chilled semen, Synchrovine® treated ewes TAI into the cervix at 51 or 57 h were similar in NRR21d (0.16 vs 0.20), Fertility (0.12 vs 0.14), and Fecundity (0.12 vs 0.15), respectively; but lower (P < 0.05) than P4-eCG treated ewes TAI at 54 h (0.34, 0.28, and 0.33 for NRR21d, Fertility and Fecundity respectively). Synchrovine® treated ewes intrauterine TAI at 51 or 57 h yielded similar NRR21d (0.51 vs 0.58), Fertility (0.43 vs 0.51), and Fecundity (0.45 vs 0.56) respectively, but lower (P < 0.05) results compared to P4-eCG treated ewes (0.75, 0.71, and 0.88 for NRR21d, Fertility and Fecundity respectively). In conclusion, AI-time in Synchrovine® treated ewes with fresh semen might be extended (42 to 54 h after the second PGF(2α)), but should be delayed to 48-54 h with chilled semen and cervical AI. Independent of the seminal state, AI-time or AI-pathway, Synchrovine® yielded lower reproductive results than a conventional P4-eCG protocol.

Effects of prostaglandin administration on ovarian follicular dynamics, conception, prolificacy, and fecundity in sheep.

Two experiments were conducted to determine the effects of prostaglandin administration on ovarian follicular dynamics, conception, prolificacy, and fecundity in sheep. During the breeding season, multiparous Corriedale ewes were randomly allocated to two groups: 1) PG group (n = 15 and n = 135 in Experiments I and II, respectively): synchronized with two injections of DL-Cloprostenol (125 μg) given 7 d apart, and inseminated at a fixed time (Day 0), 48 h after the second injection; and 2) Control group (n = 15 and n = 73 in Experiments I and II): ewes in spontaneous estrus inseminated at detected estrus. Ewes received 100 × 10(6) sperm by intrauterine AI. Ultrasonography was used to evaluate growth of the ovulatory follicle, ovulation rate (OR), conception rate, and prolificacy on Days 30 and 60. Ewes from the group PG had a larger (4.8 ± 0.5 mm, mean ± SEM; P < 0.05) ovulatory follicle that grew faster (1.2 ± 0.3 mm/d, P = 0.08), and a lower OR (1.37 ± 0.1, P < 0.05), compared to ewes from the Control group (3.9 ± 0.2 mm, 0.7 ± 0.2 mm/d, and 1.61 ± 0.1 respectively). Plasma progesterone concentrations from Days -6 to 1 were lower in the PG group (P < 0.05), but plasma estradiol concentrations were similar between groups (P > 0.05). Progesterone concentrations were similar between groups during the early luteal phase and on Days 12 and 17 (P > 0.05). The embryo recovery rate (Day 7) tended to be lower in the PG group (39 vs 64%, P = 0.08), but embryo quality did not differ between groups. Conception, prolificacy and fecundity, were lower in the PG than in the Control group (P < 0.05). Cumulative reproductive losses were similar between groups, but more twins were lost in the PG group (P < 0.05). We concluded that in ewes synchronized with PGF(2α) given twice, 7 d apart, lower reproductive performance was associated with an environment dominated by lower progesterone concentrations that stimulated the preovulatory follicle to grow faster and become larger; this was associated with lower rates of ovulation, conception, prolificacy, and fecundity.

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.

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.

Chilled Storage of Ram Semen Improves with the Addition of Egg Yolk and Glycerol to Milk-Based Extenders

This study aimed at comparing in vitro, ultra-heat-treated (UHT) skim milk and INRA-96(®) -based extenders supplemented or not with 5% egg yolk and/or 2% glycerol on sperm quality parameters along 72 h of preservation at 5 °C, using a factorial design. Semen from six healthy mature Merino rams was pooled and extended in each medium using a split sample procedure (six replicates) and chilled. Subjective motility (SM) (%), membrane integrity (MI) (%) and uncapacitated spermatozoa (US) (× 10(6) spermatozoa/AI dose) were used to assess the semen quality at 0, 12, 24, 48 and 72 h of preservation. UHT-based extenders yielded better (p<0.05) SM and MI than INRA-96(®) -based extenders (59.7% vs 57.9%; 60.2% vs 55.8%, respectively) but similar numbers of US (64.2% vs 62.3 × 10(6) sperm/AI dose, respectively) along the preservation time. Egg yolk-glycerol or just egg yolk as additives improved (p<0.05) the results compared with the base extenders without additives or just with glycerol. The sperm parameters assessed decline slowly from 0 to 48 h, with a sharp decline (p<0.05) at 72 h of preservation. In conclusion, UHT and INRA-96(®) were similar as base extenders, and the addition of 5% egg yolk plus 2% glycerol or just 5% egg yolk improved the quality of ram semen preserved at 5 °C, at least for 48 h. The combination of egg yolk-glycerol might provide extra protection in case of fluctuation of temperatures below 5 °C, commonly seen under field conditions.

Protein supplementation during a short-interval prostaglandin-based protocol for timed AI in sheep.

The aim of this experiment was to improve the reproductive performance of a short-interval prostaglandin (PG)-based protocol for timed artificial insemination in sheep, using a short-term nutritional treatment. During the breeding season (March-April), 132 multiparous and 61 nulliparous Corriedale ewes grazing natural pastures (600 kg DM/ha, 8.5% CP), were allocated to two groups: 1, Control group (n=100) two injections of D-Cloprostenol (75 μg per dose, 7d apart: Synchrovine(®) protocol); and 2, Supplemented group (n=93) ewes in which stage of the oestrous cycle was synchronised with Synchrovine(®) protocol plus focus feeding of a protein supplement (33.8% CP) between PG doses (Day -7 to -2). Cervical AI was performed at fixed time (Day 0), 46 ± 1.0 h after the second PG injection using 150 million sperm per ewe. Ovulation rate (Day 10), pregnancy rate, prolificacy and fecundity at Day 69 were evaluated by ultrasonography. Ovulation rate at Day 10 (1.20 ± 0.05 vs. 1.22 ± 0.05), pregnancy (46 ± 0.05 vs. 56 ± 0.05), prolificacy (1.09 ± 0.04 vs. 1.06 ± 0.05), and fecundity (0.49 ± 0.06 vs. 0.59 ± 0.06) at Day 69, were similar between groups (P>0.05; Control and Supplemented group respectively). It is concluded that focus feeding for 6d with protein supplementation during a short-interval PG-based protocol (Synchrovine(®)) did not improve the reproductive outcome associated with this protocol.

Conception rates in ewes after AI with ram semen preserved in milk-egg yolk extenders supplemented with glycerol.

This study aimed at comparing the effect of ram semen preserved at 5 °C on two milk-based extenders (UHT skim milk or INRA-96(®) , 5% egg yolk) supplemented with 2% glycerol, and the preservation time (24 and 48 h) on conception rates after cervical AI of ewes. In two field trials, 1198 Merino ewes were cervical AI in spontaneous oestrus. In Experiment 1, pooled semen (6 rams) was extended in UHT-base (fresh, control) or chilled for 24 h in UHT5Y (UHT-base 5% egg yolk), INRA5Y (INRA-96(®) 5% egg yolk), UHT5Y2G (UHT5Y 2% glycerol) or INRA5Y2G (INRA5Y 2% glycerol). In Experiment 2, AI was performed with pooled semen (7 rams) used fresh (extended in UHT-base or UHT5Y2G, control groups) or chilled (extended in UHT5Y2G) for 24 or 48 h. Conception rate was determined by ultrasound 40 days after AI. INRA-96(®) - had similar conception as UHT-preserved semen (56.7 vs 55.4%, p>0.05). Addition of 2% glycerol did not modify the results (56.8 vs 55.2%, p>0.05). Fresh semen extended in UHT-base, and UHT5Y2G yielded similar conception rates (60 vs 64%, p>0.05). Preservation for 24 or 48 h in UHT5Y2G gave similar results (49 vs 47%; p>0.05). In conclusion, ram semen chilled for 24 h in UHT- or INRA-96(®) -based extenders yielded similar results, and glycerol addition did not have a detrimental effect. UHT5Y2G might be used to extend ram semen for fresh AI, or to preserve it for 24 or 48 h with acceptable results.