A. Garcia-Guerra

Trio, a novel high fecundity allele: I. Transcriptome analysis of granulosa cells from carriers and noncarriers of a major gene for bovine ovulation rate

Abstract A major gene for bovine ovulation rate has been mapped to a 1.2 Mb region of chromosome 10. Screening of coding regions of positional candidate genes within this region failed to reveal a causative polymorphism, leading to the hypothesis that the phenotype results from differences in candidate gene expression rather than alteration of gene structure. This study tested differences in expression of positional candidate genes in granulosa cells between carriers and noncarriers of the high fecundity allele, as well as characterizing differences in the transcriptomic profile between genotypes. Five carriers and five noncarriers, female descendants of “Trio,” a carrier of the high fecundity allele were initially used in an RNA-seq analysis of gene expression. Four of ten samples were contaminated with theca cells, so that six samples were used in the final analysis (three of each genotype). Of 14 973 genes expressed, 143 were differentially expressed (false discovery rate P < 0.05) in carriers versus noncarriers. Among the positional candidate genes, SMAD6 was 6.6-fold overexpressed in the carriers compared to noncarriers (P < 5 × 10-5). This result was replicated in an independent group of 12 females (7 carriers and 5 noncarriers) using quantitative real-time PCR; SMAD6 was 9.3-fold overexpressed in carriers versus noncarriers (P = 1.17 × 10-6). Association of overexpression of SMAD6, an inhibitor of the BMP/SMAD signaling pathway, with high ovulation rate corresponds well with disabling mutations in ligands (BMP15 and GDF9) and a receptor (BMPR1B) of this pathway that cause increased ovulation rate in sheep. Summary Sentence Carriers of an allele for high ovulation rate exhibit a six-fold overexpression of the positional candidate gene SMAD6 compared to noncarriers, strongly implicating SMAD6 as the gene responsible for the high ovulation rate phenotype.

Trio, a novel bovine high fecundity allele: III. Acquisition of dominance and ovulatory capacity at a smaller follicle size

Abstract The acquisition of dominance and ovulatory capacity was evaluated in follicles from cows that were carriers or half-sibling noncarriers of the Trio allele. Follicle size at acquisition of follicular dominance was determined by evaluating whether follicles ovulate after GnRH challenge (ovulatory capacity—experiment 1) and by determination of intrafollicular concentrations of estradiol and free insulin like growth factor 1 (IGF1) and relative mRNA expression of cytochrome P450 family 19 subfamily A member 1 (CYP19A1), luteinizing hormone/choriogonadotropin receptor (LHCGR), and pappalysin 1 (PAPPA, previously known as pregnancy-associated plasma protein A, pappalysin 1) in granulosa cells from follicles of different sizes (experiment 2). Ovulatory capacity developed in follicles at 8.3 mm (50% ovulatory capacity) in noncarriers but at smaller sizes (5.5 mm) in Trio carriers. Similarly, in experiment 2, follicles of Trio carriers acquired a dominant phenotype, as determined by intrafollicular estradiol and CYP19A1, LHCGR, and PAPPA mRNA expression in granulosa cells, at significantly smaller sizes but at a similar time after wave emergence. Overall, dominance/ovulatory capacity was acquired when follicles of Trio carriers were ∼30% the size (volume basis) of follicles in noncarriers. In addition, follicles in Trio carriers appear to acquire dominance in a hierarchal manner, as demonstrated by the progressively greater number of follicles with a dominant phenotype between days 2 and 4 after wave emergence. Thus, results from this study provide further support for a physiological model in which selection of multiple follicles in Trio allele carriers is characterized by acquisition of dominance at a smaller follicle size but at a similar time in the follicular wave with multiple follicles acquiring dominance in a hierarchal sequence. Summary Sentence Results support a model for selection of multiple follicles in Trio allele carriers due to acquisition of dominance at a smaller follicle size but similar time in the follicular wave with multiple follicles acquiring dominance in a hierarchal sequence.

Effect of feeding rumen-protected methionine on productive and reproductive performance of dairy cows

The objectives of this study were to evaluate the effects of daily top-dressing (individually feeding on the top of the total mixed ration) with rumen-protected methionine (RPM) from 30 ± 3 until 126 ± 3 Days in milk on productive and reproductive performance in lactating dairy cows. A total of 309 lactating dairy Holstein cows (138 primiparous and 171 multiparous) were randomly assigned to treatment diets containing either RPM (21.2 g of RPM + 38.8 g of dried distillers grain; 2.34% Methionine [Met] of metabolizable protein [MP]) or Control (CON; 60 g of dried distillers grain; 1.87% Met of MP). Plasma amino acids were evaluated at the time of artificial insemination (AI) and near pregnancy diagnosis. Milk production and milk composition were evaluated monthly. Pregnancy was diagnosed on Day 28 (by Pregnancy-specific protein B [PSPB]), 32, 47, and 61 (by ultrasound) and sizes of embryonic and amniotic vesicle were determined by ultrasound on Day 33 after AI. Feeding RPM increased plasma Met at 6, 9, 12, and 18 hours after top-dressing with a peak at 12 hours (52.4 vs 26.0 μM; P < 0.001) and returned to basal by 24 hours. Cows fed RPM had a small increase in milk protein percentage (3.08 vs 3.00%; P = 0.04) with no differences on milk yield and milk protein yield. Additionally, in multiparous cows, RPM feeding increased milk protein (3.03 vs 2.95%; P = 0.05) and fat (3.45 vs 3.14%; P = 0.01) percentages, although no effects were observed in primiparous cows. In multiparous cows fed RPM, pregnancy loss was lower between Days 28 to 61 (19.6 [10/51] vs. 6.1% [3/49]; P = 0.03) or between Days 32 to 61 (8.9 [4/45] vs. 0 [0/0] %; P = 0.03), although, there was no effect of treatment on pregnancy loss in primiparous cows. Consistent with data on pregnancy loss, RPM feeding increased embryonic abdominal diameter (P = 0.01) and volume (P = 0.009) and amniotic vesicle volume (P = 0.04) on Day 33 of pregnancy in multiparous cows but had no effect on embryonic size in primiparous cows. Thus, the increase in plasma Met concentrations after feeding RPM was sufficient to produce a small increase in milk protein percentage and to improve embryonic size and pregnancy maintenance in multiparous cows. Further studies are needed to confirm these responses and understand the biological mechanisms that underlie these responses as well as the timing and concentrations of circulating Met that are needed to produce this effect.

101 DOSE AND TIMING OF ADMINISTRATION OF PROSTAGLANDIN F2α DURING FIXED-TIME EMBRYO TRANSFER IN AN IN VITRO-PRODUCTION PROGRAM

Synchronization protocols for fixed-time embryo transfer (ET) contribute significantly to the overall cost of an in vitro-produced-ET program, primarily through the cost of drugs and the labour required. Optimization of synchronization protocols to reduce cost, while providing high fertility, have the potential to improve overall efficiency and profitability. The objective of the present study was to evaluate the effect of dose and schedule of administration of prostaglandin F2α (PGF) during a synchronization protocol for fixed-time ET. Holstein and cross-bred Holstein heifers (n=3766) were synchronized using a modified 5-day CIDR Synch as follows: Day 0: CIDR inserted; Day 5: CIDR removed, PGF2α treatment; Day 8: gonadotropin-releasing hormone (GnRH; 100μg of gonadorelin). On Day 5, at the time of CIDR removal, heifers were randomly assigned to a 2×2 factorial design to receive either a full or half dose of PGF (Cloprostenol; 500v. 250μg) and 1 (Day 5) or 2 (Day 6) administrations resulting in the following treatments: full dose Day 5+Day 6 (n=938); full dose Day 5 (n=938); half dose Day 5+Day 6 (n=946); and half dose Day 5 (n=944). Heifers were evaluated by ultrasonography 5 days after GnRH to determine presence and size of the corpus luteum. Heifers with a corpus luteum received a fresh in vitro-produced embryo 7±1 days after GnRH administration, and pregnancy was determined by ultrasonography 32 and 60 days after GnRH. Fertility data were analysed by logistic regression and included the fixed effects of dose, time, and their interaction. Fertility results are shown in Table 1. Utilisation rate (transferred/treated) was not affected by dose (P=0.66), time (P=0.19), or their interaction (P=0.17). The percentage of heifers detected in oestrus was not affected by dose (P=0.13), time (P=0.72), or their interaction (P=0.89). There were no significant differences between doses of PGF (P=0.32), time (P=0.71), or their interaction (P=0.80) on pregnancies per ET on Day 32. Similarly, no differences were found on pregnancies per ET on Day 60 between doses (P=0.35), time (P=0.96), or their interaction (P=0.89). In addition, pregnancy loss between Day 32 and 60 was not affected by dose (P=0.76), time (P=0.66), or their interaction (P=0.54). In conclusion, the use of a half dose of PGF once on Day 5 resulted in comparable utilisation rate and fertility as the observed with 2 full dose applications 24h apart. As a result, the overall cost of the fixed-time ET program can be reduced by eliminating the need for a second PGF treatment and by decreasing the dose without compromising fertility.

102 UNILATERAL AND BILATERAL TRANSFER OF 2 IN VITRO-PRODUCED EMBRYOS INCREASES PREGNANCY LOSS BETWEEN 30 AND 60 DAYS.

Fertility of in vitro-produced embryos is affected by embryo stage and quality. Embryos quality 1 and stage 7 result in higher fertility than embryos of earlier stages and/or lower quality. The objective was to evaluate the effect of unilateral and bilateral transfer of 2in vitro-produced embryos of earlier stages and/or poor quality on fertility. Heifers were synchronized using a 5-day CIDR Synch or 2 prostaglandin F2α injections 14 days apart followed by oestrus detection. Embryo transfer was performed 7±1 day after gonadotropin-releasing hormone/oestrus and heifers were assigned randomly to 1 of 3 groups: single embryo ipsilateral to the corpus luteum (single; n=188); 2 embryos in the uterine horn ipsilateral to the corpus luteum (unilateral; n=138); 2 embryos bilaterally (bilateral; n=128). Embryos stage 4 to 8 and quality 1 or 2 were randomly assigned to treatment groups. All embryos were 7-day fresh in vitro-produced embryos, and pregnancy diagnosis was performed by ultrasonography on Days 32 and 60. Data were analysed by logistic regression. Conception rates on Days 32 and 60 were not different (P>0.10) between heifers receiving a single embryo [Day 32=30.9% (58/188) and Day 60=25% (47/188)] or those receiving 2 embryos [Day 32=36.5% (97/266) and Day 60=22.2% (59/266)]. However, pregnancy loss between Days 32 and 60 was greater (P<0.01) in heifers with 2 embryos (39.2%; 38/97) than in those with a single embryo (18.9%; 11/58). Conception rate on Day 32 was not different between groups (P=0.4) and was 30.9% (58/188) for single, 36.9% (51/138) for unilateral, and 35.9% (46/128) for bilateral. Similarly, there was no difference (P=0.8) in conception rates on Day 60, single=25% (47/188), unilateral=23.9% (33/138), and bilateral=20.3% (26/128). However, there was an effect of group on pregnancy loss between Days 32 and 60 (P=0.04). Losses were higher (P=0.01) in the bilateral group [43.5% (20/46)] compared with the single group [18.9% (11/58)], and the unilateral group was intermediate [35.3% (18/51)] and tended to be different from the single group (P=0.1). Interestingly, when pregnancy loss was compared between heifers with twin or single pregnancies, as determined by ultrasonography, and regardless of the number of embryos transferred or their location, twin pregnancies had a greater pregnancy loss [62.1% (18/29)] compared with single pregnancies [24.6% (31/126); P<0.01]. For twin bearing heifers, as determined by ultrasonography on Day 32, pregnancy loss did not differ between unilateral (62.5%; 10/16) and bilateral (61.5%; 8/13) transfers (P=0.9). Similarly, there was no difference (P=0.2) for heifers with single embryo pregnancies: single (18.9%; 11/58), unilateral (22.9%; 8/35), bilateral (36.4%; 12/33), although bilateral transfer of 2 embryos tended to be higher than single (P=0.07). Transfer of 2 low quality in vitro-produced embryos results in similar conception rates, although pregnancy losses are greater. Interestingly, only 30% (29/97) of the pregnancies from heifers that received 2 embryos contained twins, indicating the loss of one of the embryos before Day 32. Furthermore, the increased losses observed with the transfer of 2 embryos were attributed to those heifers in which twin pregnancies were diagnosed on Day 32 regardless of distribution.