R.W. Bender

Improving fertility to timed artificial insemination by manipulation of circulating progesterone concentrations in lactating dairy cattle

This manuscript reviews the effect of progesterone (P4) during timed AI protocols in lactating dairy cows. Circulating P4 is determined by a balance between P4 production, primarily by the corpus luteum (CL), and P4 metabolism, primarily by the liver. In dairy cattle, the volume of luteal tissue is a primary determinant of P4 production; however, inadequate circulating P4 is generally due to high P4 metabolism resulting from extremely elevated liver blood flow. Three sections in this manuscript summarise the role of P4 concentrations before breeding, near the time of breeding and after breeding. During timed AI protocols, elevations in P4 are generally achieved by ovulation, resulting in an accessory CL, or by supplementation with exogenous P4. Elevating P4 before timed AI has been found to decrease double ovulation and increase fertility to the timed AI. Slight elevations in circulating P4 can dramatically reduce fertility, with inadequate luteolysis to the prostaglandin F2α treatment before timed AI being the underlying cause of this problem. After AI, circulating P4 is critical for embryo growth, and for establishment and maintenance of pregnancy. Many studies have attempted to improve fertility by elevating P4 after timed AI with marginal elevations in fertility. Thus, previous research has provided substantial insights into mechanisms regulating circulating P4 concentrations and actions. Understanding this prior research can focus future research on P4 manipulation to improve timed AI protocols.

Patterns of Gene Expression in the Bovine Corpus Luteum Following Repeated Intrauterine Infusions of Low Doses of Prostaglandin F2alpha

ABSTRACT Natural luteolysis involves multiple pulses of prostaglandin F2alpha (PGF) released by the nonpregnant uterus. This study investigated expression of 18 genes from five distinct pathways, following multiple low-dose pulses of PGF. Cows on Day 9 of the estrous cycle received four intrauterine infusions of 0.25 ml of phosphate-buffered saline (PBS) or PGF (0.5 mg of PGF in 0.25 ml of PBS) at 6-h intervals. A luteal biopsy sample was collected 30 min after each PBS or PGF infusion. There were four treatment groups: Control (n = 5; 4 PBS infusions), 4XPGF (4 PGF infusions; n = 5), 2XPGF-non-regressed (2 PGF infusions; n = 5; PGF-PBS-PGF-PBS; no regression after treatments), and 2XPGF-regressed (PGF-PBS-PGF-PBS; regression after treatments; n = 5). As expected, the first PGF pulse increased mRNA for the immediate early genes JUN, FOS, NR4A1, and EGR1 but unexpectedly also increased mRNA for steroidogenic (STAR) and angiogenic (VEGFA) pathways. The second PGF pulse induced immediate early genes and genes related to immune system activation (IL1B, FAS, FASLG, IL8). However, mRNA for VEGFA and STAR were decreased by the second PGF infusion. After the third and fourth PGF pulses, a distinctly luteolytic pattern of gene expression was evident, with inhibition of steroidogenic and angiogenic pathways, whereas, there was induction of pathways for immune system activation and production of PGF. The pattern of PGF-induced gene expression was similar in corpus luteum not destined for luteolysis (2X-non-regressed) after the first PGF pulse but was very distinct after the second PGF pulse. Thus, although the initial PGF pulse induced mRNA for many pathways, the second and later pulses of PGF appear to have set the distinct pattern of gene expression that result in luteolysis.

Effect of treatment with human chorionic gonadotropin on day 5 after timed artificial insemination on fertility of lactating dairy cows

Reproductive management programs that synchronize ovulation can ovulate a smaller than normal follicle, potentially resulting in inadequate progesterone (P4) concentrations after artificial insemination (AI). Ovulation of the dominant follicle of the first follicular wave with human chorionic gonadotropin (hCG) treatment can produce an accessory corpus luteum and increase circulating P4 concentrations. This manuscript reports the results of 2 separate analyses that evaluated the effect of hCG treatment post-AI on fertility in lactating dairy cows. The first study used meta-analysis to combine the results from 10 different published studies that used hCG treatment on d 4 to 9 post-AI in lactating dairy cows. Overall, pregnancies per artificial insemination (P/AI) were increased 3.0% by hCG treatment post-AI [34% (752/2,213) vs. 37% (808/2,184); Control vs. hCG-treated, respectively]. The second study was a field research trial in which lactating Holstein cows (n=2,979) from 6 commercial dairy herds were stratified by parity and breeding number and then randomly assigned to one of 2 groups: control (no further treatment, n=1,519) or hCG [Chorulon i.m.: 2,000 IU (in 3 of the herds) or 3,300 IU (in 3 herds); n=1,460] on d 5 after a timed AI (ovulation synchronized with Ovsynch, Presynch-Ovsynch, or Double-Ovsynch). In a subset of cows, the hCG profile and P4 changes were determined. Treatment with hCG increased P4 (4.3 vs. 5.3 ng/mL on d 12). Pregnancies per AI were greater in cows treated with hCG (40.8%; 596/1,460) than control (37.3%; 566/1,519) cows. Interestingly, an interaction among treatment and parity was observed; primiparous cows had greater P/AI after hCG (49.7%; 266/535) than controls (39.5%; 215/544). In contrast, older cows receiving hCG (35.7%; 330/925) had similar P/AI to controls (36.0%; 351/975).Thus, targeted use of hCG on d 5 after TAI enhances fertility about 3.0% (based on meta-analysis) to 3.5% (based on our field trial). Surprisingly, this fertility-enhancing effect of hCG was very large in first-lactation cows but not observed in older cows in the field study. Future research is needed to confirm these intriguing results and to determine why older cows did not have improved fertility after hCG treatment.

Relationship between circulating anti-Müllerian hormone (AMH) and superovulatory response of high-producing dairy cows

The main objective of this study was to evaluate the relationship between circulating anti-Müllerian hormone (AMH) and superovulatory response of dairy cows. Holstein cows (n=72) were milked twice daily and housed and fed individually in tiestalls. All animals were synchronized and flushed at 70±3 d in milk (DIM), near peak production (39.6kg/d). Blood samples for AMH analysis were collected at 3 different stages of a synchronized estrous cycle [at a random stage (40±3 DIM), proestrus (50±3 DIM), and diestrus (57±3 DIM)]. Body weights were measured weekly from calving until embryo collection. Statistical analyses were performed with Proc CORR and Proc GLIMMIX of SAS (SAS Institute Inc., Cary, NC). The 3 AMH samples from individual cows were correlated and not influenced by day of cycle. Surprisingly, AMH tended to be negatively correlated with body weight loss from calving to embryo collection (r=-0.22). More importantly, average AMH was highly associated (r=0.65) with superovulation response (number of corpora lutea on the day of the flush, CLN), total structures collected (r=0.48), and total transferable embryos (r=0.37), but not percentage of fertilized embryos (r=-0.20) or degenerate embryos (r=0.02). When cows were classified into quartiles (Q) of circulating AMH (Q1=0.01 to 82.6pg/mL; Q2=91.1 to 132.5pg/mL; Q3=135.3 to 183.8pg/mL; Q4=184.4 to 374.3pg/mL), we observed a >2-fold difference between first and fourth AMH quartiles in superovulation response (CLN: Q1=12.0±1.5; Q2=14.7±2.0; Q3=17.2±1.2; Q4=25.6±1.5) and embryo production. In conclusion, circulating AMH concentration was strongly associated with superovulation response, and evaluation of AMH could be used to identify cows with greater responses to superstimulation and thus improve efficiency of superovulation programs in dairy cows.

Effect of feed restriction on reproductive and metabolic hormones in dairy cows.

The objective of this trial was to evaluate the effects of feed restriction (FR) on serum glucose, nonesterified fatty acids, progesterone (P4), insulin, and milk production in dairy cows. Eight multiparous Holstein cows, 114 ± 14 d pregnant and 685 ± 39 kg of body weight, were randomly assigned to a replicated 4 × 4 Latin square design with 14-d periods. During the first 8 d of each period, cows in all treatments were fed for ad libitum feed intake. Beginning on d 9 of each period, cows received 1 of 4 treatments: ad libitum (AL), 25% feed restriction (25 FR), 50% feed restriction (50 FR), and 50% of TMR replaced with wheat straw (50 ST). Daily feed allowance was divided into 3 equal portions allocated every 8h with jugular blood samples collected immediately before each feeding through d 14. In addition, on d 12 of each period, blood samples were collected before and at 60, 120, 180, 240, 300, 360, 420, and 480 min after morning feeding. The conventional total mixed ration and total mixed ration with straw averaged 15.1 and 10.8%, 32.1 and 50.5%, and 26.8 and 17.0% for concentrations of crude protein, neutral detergent fiber, and starch, respectively. Cows that were feed and energy restricted had reduced dry matter intake, net energy for lactation intake, circulating glucose concentrations, and milk production, but greater body weight and body condition score losses than AL cows. Circulating concentrations of insulin were lower for cows fed 50 FR (8.27 μIU/mL) and 50 ST (6.24 μIU/mL) compared with cows fed AL (16.65 μIU/mL) and 25 FR (11.16 μIU/mL). Furthermore, the greatest plasma nonesterified fatty acids concentration was observed for 50 ST (647.7 μ Eq/L), followed by 50 FR (357.5 μEq/L), 25 FR (225.3 μEq/L), and AL (156.3 μEq/L). In addition, serum P4 concentration was lower for cows fed AL than cows fed 50 ST and 25 FR. Thus, FR reduced circulating glucose and insulin but increased P4 concentration, changes that may be positive in reproductive management programs.

Effects of acute feed restriction combined with targeted use of increasing luteinizing hormone content of follicle-stimulating hormone preparations on ovarian superstimulation, fertilization, and embryo quality in lactating dairy cows

Multiple metabolic and hormonal factors can affect the success of protocols for ovarian superstimulation. In this study, the effect of acute feed restriction and increased LH content in the superstimulatory FSH preparation on numbers of ovulations, fertilization, and embryo quality in lactating dairy cows was evaluated. Two experiments were performed using a Latin square design with treatments arranged as a 2 × 2 factorial: feed restriction (FR; 25% reduction in dry matter intake) compared with ad libitum (AL) feeding, combined with high (H) versus low (L) LH in the last 4 injections of the superstimulatory protocol. As expected, FR decreased circulating insulin concentrations (26.7 vs. 46.0 μU/mL). Two analyses were performed: one that evaluated the complete Latin square in experiment 2 and a second that evaluated only the first periods of experiments 1 and 2. For both analyses, follicle numbers, ovulation rates, and corpora lutea on d 7 were not different. In the first period analysis of experiments 1 and 2, we observed an interaction between feed allowance and amount of LH on fertilization rates, percentage of embryos or oocytes that were quality 1 and 2 embryos, and number of embryos or oocytes that were degenerate. Fertilization rates were greater for the AL-L (89.4%) and FR-H (80.1%) treatments compared with the AL-H (47.9%) and FR-L (59.9%) treatments. Similarly, the proportion of total embryos or oocytes designated as quality 1 and 2 embryos was greater for AL-L (76.7%) and FR-H (73.4%) treatments compared with AL-H (35.6%) and FR-L (47.3%) treatments. In addition, the number of degenerate embryos was decreased for AL-L (1.3) and FR-H (0.4) treatments compared with the AL-H (2.6) and FR-L (2.3) treatments. Thus, cows with either too low (FR-L) or too high (AL-H) insulin and LH stimulation had lesser embryo production after superstimulation because of reduced fertilization rate and increased percentage of degenerate embryos. Therefore, interaction of the gonadotropin content of the superstimulatory preparation with the nutritional program of the donor cow needs to be considered to optimize success of ovarian superstimulatory protocols.

Use of a single injection of long-acting recombinant bovine FSH to superovulate Holstein heifers: A preliminary study

Our objective was to compare several experimental preparations of a single injection of long-acting recombinant bovine FSH (rbFSH; types A and B) to a porcine pituitary-derived FSH (Folltropin) to superovulate Holstein dairy heifers. Nonlactating, nonpregnant virgin Holstein heifers (n = 56) aged 12 to 15 months were randomly assigned to one of four superstimulatory treatments. Beginning at a random stage of the estrous cycle, all follicles greater than 5 mm were aspirated. Thirty-six hours later, heifers received an intravaginal P4 device and superstimulatory treatments were initiated. Treatments were (1) 300 mg of pituitary-derived FSH (Folltropin) administered in eight decreasing doses over a period of 3.5 days; (2) a single injection of 50 μg of A-rbFSH; (3) a single injection of 100 μg of A-rbFSH; and (4) a single injection of 50 μg of B-rbFSH. All heifers received 25 mg PGF2α at 48 and 72 hours after the insertion of P4 device. At 84 hours after insertion, P4 devices were removed, and ovulation was induced 24 hours later with hCG (2500 IU). Heifers were inseminated at 12 and 24 hours after hCG treatment. The number of ovulatory follicles was greatest for heifers treated with Folltropin and B50-rbFSH, least for heifers treated with A50-rbFSH, and was intermediate for heifers treated with A100-rbFSH (25.7 ± 3.2, 18.9 ± 3.2, 5.9 ± 0.9, and 16.6 ± 3.1, respectively; P < 0.001). The number of corpora lutea was greatest for heifers treated with Folltropin, B50-rbFSH, and A100-rbFSH, and least for heifers treated with A50-rbFSH (19.1 ± 2.4, 16.1 ± 3.0, 15.9 ± 2.9, and 2.6 ± 0.9, respectively; P < 0.001). The number of good-quality embryos differed among treatments and was greatest for heifers treated with B50-rbFSH, Folltropin, and A100-rbFSH and least for heifers treated with A50-rbFSH (7.6 ± 2.4, 6.5 ± 1.7, 4.3 ± 1.5, and 0.8 ± 0.5, respectively; P < 0.001). In conclusion, a single injection of a preparation of long-acting rbFSH (either 100 μg of A-rbFSH or 50 μg of B-rbFSH but not 50 μg of A-rbFSH) produced similar superovulatory responses resulting in the production of good-quality embryos when compared with a pituitary-derived FSH preparation administered twice daily for 4 days. More studies using different types of cattle and different doses of rbFSH are needed to confirm the findings reported in this preliminary study.

Comparison of in situ versus in vitro methods of fiber digestion at 120 and 288 hours to quantify the indigestible neutral detergent fiber fraction of corn silage samples.

Ruminal digestion of neutral detergent fiber (NDF) is affected in part by the proportion of NDF that is indigestible (iNDF), and the rate at which the potentially digestible NDF (pdNDF) is digested. Indigestible NDF in forages is commonly determined as the NDF residue remaining after long-term in situ or in vitro incubations. Rate of pdNDF digestion can be determined by measuring the degradation of NDF in ruminal in vitro or in situ incubations at multiple time points, and fitting the change in residual pdNDF by time with log-transformed linear first order or nonlinear mathematical treatments. The estimate of indigestible fiber is important because it sets the pool size of potentially digestible fiber, which in turn affects the estimate of the proportion of potentially digestible fiber remaining in the time series analysis. Our objective was to compare estimates of iNDF based on in vitro (IV) and in situ (IS) measurements at 2 fermentation end points (120 and 288h). Further objectives were to compare the subsequent rate, lag, and estimated total-tract NDF digestibility (TTNDFD) when iNDF from each method was used with a 7 time point in vitro incubation of NDF to model fiber digestion. Thirteen corn silage samples were dried and ground through a 1-mm screen in a Wiley mill. A 2×2 factorial trial was conducted to determine the effect of time of incubation and method of iNDF analysis on iNDF concentration; the 2 factors were method of iNDF analysis (IS vs. IV) and incubation time (120 vs. 288h). Four sample replicates were used, and approximately 0.5g/sample was weighed into each Ankom F 0285 bag (Ankom Technology, Macedon, NY; pore size=25 µm) for all techniques. The IV-120 had a higher estimate of iNDF (37.8% of NDF) than IS-120 (32.1% of NDF), IV-288 (31.2% of NDF), or IS-288 technique (25.7% of NDF). Each of the estimates of iNDF was then used to calculate the rate of degradation of pdNDF from a 7 time point in vitro incubation. When the IV-120 NDF residue was used, the subsequent rates of pdNDF digestion were fastest (2.8% h(-1)) but the estimate of lag was longest (10.3h), compared with when iNDF was based on the IS-120 or IV-288 NDF residues (rates of 2.3%h(-1) and 2.4%h(-1); lag times of 9.7 and 9.8 h, respectively). Rate of pdNDF degradation was slowest (2.1% h(-1)) when IS-288 NDF residue was used as the estimate of iNDF. The estimate of lag based on IS-288 (9.4h) was similar to lag estimates calculated when IS-120 or IV-288 were used as the estimate of iNDF. The TTNDFD estimates did not differ between treatments (35.5%), however, because differences in estimated pools of iNDF resulted in subsequent changes in rates and lag times of fiber digestion that tended to cancel out. Estimates of fiber digestion kinetic parameters and TTNDFD were similar when fit to either the linear or nonlinear fiber degradation models. All techniques also yielded estimates of iNDF that were higher than predicted iNDF based on the commonly used ratio of 2.4 × lignin.

The effects of calcium hydroxide–treated whole-plant and fractionated corn silage on intake, digestion, and lactation performance in dairy cows

The objective of this trial was to evaluate, in dairy cattle, the effects of calcium hydroxide treatment of whole-plant corn and a treatment applied to the bottom stalk fraction of the corn plant, achieved by harvesting corn in 2 crop streams. The treatments were calcium hydroxide-treated corn silage (TRTCS), toplage supplemented with calcium hydroxide-treated stalklage (TPL), a positive control of brown midrib corn silage (BMR), and a negative control of conventional whole-plant corn silage (WPCS). The toplage was harvested at a height of 82 cm with 2 of the 6 rows set as ear-snapping to incorporate higher tissues into the stalklage. Stalklage was harvested at 12 cm, and other corn silages were harvested at 27 cm. Sixteen pens, each with 8 Holstein cows averaging 70±25 d in milk and 46±11 kg of milk d(-1), were assigned 4 per treatment in a completely randomized design. The diet was approximately 40% corn silage, 20% alfalfa silage, and 40% concentrate on a dry matter basis. A 2-wk covariate period with conventional corn silage was followed by an 8-wk treatment period in which the 4 corn silage treatments were the only effective difference in diets. Cows fed TPL and TRTCS consumed more (1.9 and 1.4 kg of organic matter d(-1), respectively) than did cows fed WPCS. Milk yield was greater for cows fed BMR, TPL, and TRTCS. Cows fed BMR and TPL produced 2.9 and 2.7 kg d(-1), respectively, more energy-corrected milk (ECM) than cows fed WPCS, and cows fed TRTCS had the greatest ECM production (4.8 kg of ECM d(-1) greater than cows fed WPCS). No differences in body weight or body condition scored were observed. Milk fat concentration was similar among treatments and milk protein concentration was reduced for TRTCS. Starch and neutral detergent fiber digestibility were greater for cows fed TRTCS.

Effects of propylene glycol or elevated luteinizing hormone during follicle development on ovulation, fertilization, and early embryo development†

Abstract Seventeen nonlactating Holstein cows were superovulated in a Latin-square designed experiment to determine the effects of increased propylene glycol (PROP) and luteinizing hormone (LH) during antral follicle development on ovarian function, fertilization, and early embryo quality. PROP was orally drenched every 4 h for 7 days to induce hyperinsulinemia and associated metabolic changes. LH concentrations were altered by increasing LH (3-fold) during last 2 days of superovulation. Treatment groups were as follows: (1) control—oral drenching with water plus low-LH preparation; (2) high LH(HLH)—water plus HLH preparation; (3) PROP—drenching with PROP plus low LH; (4) PROP/HLH—PROP plus HLH. PROP increased glucose (P < 0.05) and insulin (P < 0.02) concentrations at all time points analyzed. Neither PROP nor LH affected numbers of follicles > 9 mm at time of gonadotropin-releasing hormone-induced LH surge, although percentage of these follicles that ovulated was decreased by both PROP (P = 0.002) and LH (P = 0.048). In addition, PROP tended (P = 0.056) to decrease total number of ovulations. PROP reduced (P = 0.028) fertilization rate, while LH tended (P = 0.092) to increase fertilization rate. There was no effect of either PROP or LH on any measure of embryo quality including percentage of embryos that were degenerate, quality 1, or quality 1 and 2 of total structures collected or fertilized structures. These results indicate that acute elevation in insulin during the preovulatory follicular wave can decrease percentage of large follicles that ovulate, particularly when combined with increased LH, and reduce fertilization of ovulated oocytes. Summary Sentence High circulating insulin, due to treatment with propylene glycol every 4 h during final week before ovulation, reduced risk of ovulation of large follicles, reduced fertilization of ovulated oocytes, but had no effect on later embryo development.