Profiles of interferon-stimulated genes in multiple tissues and circulating pregnancy-associated glycoproteins and their association with pregnancy loss in dairy cows†.

Pregnancy loss (PL) in lactating dairy cows disrupts reproductive and productive efficiency. We evaluated the expression of interferon-stimulated genes (ISG) in blood leukocytes, vaginal and cervical epithelial cells, luteolysis-related genes, progesterone, and pregnancy-associated glycoprotein (PAG) profiles in lactating dairy cows (n = 86) to gain insight about PL. Expression of ISG on d17, d19, and d21 was greater in cows that maintained the pregnancy (P33) compared to nonpregnant with no PL (NP). Greater ISG differences between groups were observed in the cervix (96.7-fold) than vagina (31.0-fold), and least in blood leukocytes (5.6-fold). Based on individual profiles of ISG and PAG, PL was determined to occur either before (~13%) or after (~25%) d22. For cows with PL before d22, ISG expression was similar on d17 but by d21 was lower and OXTR was greater than P33 cows and similar to NP; timing of luteolysis was similar compared to NP cows suggesting embryonic failure to promote luteal maintenance and to attach to the endometrium (no increase in PAG). For cows with PL after d22, ISG expression was similar to P33 cows on d17, d19, and d21 and luteolysis, when it occurred, was later than NP cows; delayed increase in PAG suggested later or inadequate embryonic attachment. In conclusion, PL before d22 occurred due to embryonic demise/failure to signal for luteal maintenance, as reflected in reduced ISG expression by d21. Alternatively, embryos with PL between d22 and 33 adequately signaled for luteal maintenance (ISG) but had delayed/inadequate embryonic attachment and/or inappropriate luteolysis causing PL.

Effect of 200 µg of gonadorelin at the first gonadotropin-releasing hormone of the Resynch-25 on ovarian dynamics and fertility in lactating Holstein cows.

Our objective was to determine the effect of a 200-µg dose of GnRH 25 d after previous artificial insemination (AI) in a Resynch-25 resynchronization program on ovulatory response, circulating progesterone (P4) concentrations before and after treatment, and pregnancy per AI (P/AI) compared with a 100-µg dose in lactating Holstein cows. Experimental d 0 was considered the day of the previous AI. Lactating dairy cows (n = 3,240) with an average of 126 d in milk (DIM) and between 1 and 6 services were randomly assigned to receive 100 µg or 200 µg of GnRH on d 25 (GnRH25). On d 32 after AI, cows diagnosed nonpregnant with the presence of a corpus luteum (CL) detected by ultrasound (n = 1,249) received PGF2α treatments on d 32 and 33, followed by a GnRH 32 h later and AI 16 h after this last GnRH. Blood samples were collected on d 25, 32, and 34 to evaluate serum P4 concentrations. Transrectal ultrasonographic examination was performed on d 25 and 27 to assess ovulatory response to GnRH25. Cows were checked for pregnancy on d 32, 46, and 88 after AI. The larger dose of GnRH increased the overall proportion of cows that ovulated to the GnRH25 (25.0% for the 100-µg dose vs. 32.5% for the 200-µg dose). However, when cows were evaluated separately according to the pregnancy status on d 32 after AI, we found no treatment effect within cows pregnant and nonpregnant. Even though treatment increased the proportion of cows with serum P4 ≤0.42 ng/mL at the last GnRH treatment (G2; 86.2% for the 100-µg dose vs. 93.0% for the 200-µg dose), it did not affect P/AI on d 32, 46, and 88. Furthermore, a greater proportion of cows without a functional CL at GnRH25 had circulating P4 concentrations ≥1.00 ng/mL on d 32 and lower than 0.42 ng/mL on G2. These cows also had a greater P/AI on d 32, 46, and 88. In summary, the larger dose of GnRH on d 25 after AI did not increase the ovulatory response in nonpregnant cows and P/AI on d 32, 46, and 88 after AI after the Resynch-25 program. Additionally, nonpregnant cows without a functional CL at GnRH25 were better synchronized after the Resynch-25 protocol and had greater P/AI on d 32, 46, and 88 after timed-AI.

Ex vivo study shows novel, rapid, suture-free tenotomy technique for the equine deep digital flexor tendon.

OBJECTIVE: To describe the feasibility of a novel thread-transecting technique for the tenotomy of the equine deep digital flexor tendon (DDFT). ANIMALS: 39 equine distal limb specimens. METHODS: Under ultrasonographic guidance, a surgical thread was percutaneously placed around the DDFT through 2 needle punctures (lateral and medial) using a Tuohy needle in equine limbs (22 forelimbs, 17 hindlimbs). The DDFT was transected by a back-and-forth motion of the thread until the loop emerged from the entry puncture site. Each specimen was dissected and assessed for completeness of transection and iatrogenic damage under direct visualization. Descriptive statistics were reported. RESULTS: Complete DDFT transection was achieved in all 39 limbs, taking an average of 8.6 minutes per procedure. Iatrogenic damage to surrounding structures occurred in 17 (44%) limbs, with 6 (15%) limbs having more than 1 structure damaged. Damage to the communicating branch of the palmar or plantar nerves was the most commonly seen. CLINICAL RELEVANCE: DDFT tenotomy in equine limb specimens was effectively performed using a novel thread-transecting technique. The procedure is quick, and no suturing is needed, but damage to surrounding structures is possible. Further assessment of the procedure and clinical significance of its potential iatrogenic damage in clinical cases is needed.

Effect of inducing accessory corpus luteum formation with gonadotropin-releasing hormone or human chorionic gonadotropin on the day of embryo transfer on fertility of recipient dairy heifers and lactating cows.

The objective was to determine the effect of inducing an accessory corpus luteum (CL) using GnRH or human chorionic gonadotropin (hCG) on the day of in vitro-produced (IVP) embryo transfer (ET) on pregnancy per ET (P/ET) and calving/ET in dairy heifers and lactating cows. Dairy heifers (11-15 mo of age; n = 1,547) and lactating cows (n = 1,480) detected in estrus by tail chalk (d 0) were used as recipients. Before ET, the presence of a CL was evaluated by transrectal palpation from d 6 to 9 of the estrous cycle. Animals with a CL were randomly assigned to receive 1 of 3 treatments immediately before ET: control (no treatment; n = 1,009), GnRH (86 µg of GnRH; n = 1,085) and hCG (2,500 IU; n = 1,069). Embryos were implanted in the uterine horn ipsilateral to the ovary with a CL (fresh IVP embryos, n = 2,544; vitrified IVP embryos n = 545; slow-freezing IVP embryos, n = 74). Pregnancy diagnosis was performed on d 37 ± 3 of gestation by transrectal palpation. Pregnancy loss data and calving records were collected from the dairy farm management software. Treatment did not affect P/ET, calving/ET, or pregnancy loss either overall or within parity. When treatments inducing CL formation were combined (GnRH + hCG), heifers tended to have greater P/ET than controls (67.7 vs. 63.5%, respectively). Yet, calving/ET were similar. Response variables were also analyzed within embryo type and parity. For heifers receiving stage 6 (blastocyst) fresh IVP embryos, hCG had greater P/ET than controls (74.5 vs. 51.1%, respectively). In addition, GnRH tended to have greater P/ET than controls (67.8 vs. 51.2%, respectively). However, calving/ET in heifers receiving blastocyst fresh IVP embryos was similar among treatments. When only stage 7 (expanded blastocyst) fresh IVP embryos were considered, primiparous GnRH cows had greater P/ET (59.3 vs. 47.1%) and calving/ET (48.6 vs. 38.1%) than hCG. Moreover, hCG showed decreased calving/ET compared with controls in primiparous cows transferred with expanded blastocyst fresh IVP embryos. In summary, the effects of hCG or GnRH at ET on P/ET and calving/ET were inconsistent according to different embryo characteristics (e.g., embryo stage) and parity of recipients. Furthermore, treatment did not improve the overall fertility outcomes for recipient animals receiving IVP embryos.

Is pregnancy loss initiated by embryonic death or luteal regression? Profiles of pregnancy-associated glycoproteins during elevated progesterone and pregnancy loss.

Because progesterone (P4) is essential for pregnancy establishment and maintenance, we investigated the effect of increased concentrations of P4 on embryonic attachment and concentrations of pregnancy-associated glycoproteins (PAG). Additionally, we investigated the relationships among luteal regression, pregnancy loss, and PAG concentrations in cows undergoing pregnancy loss by d 33 of pregnancy. Lactating dairy cows were allocated into control (n = 40) and human chorionic gonadotropin (hCG; 3,300 IU on d 7 and 13 to promote greater circulating P4; GnRH = d 0; n = 46) groups. Progesterone was measured daily from d 7 to 33, and PAG was measured daily from d 17 to 33; both hormones were also measured on d 47 and 61. An increase in PAG >10% compared with d 17 was considered a marker for pregnancy. The gold standard for pregnancy diagnosis was ultrasound evaluation of embryonic heartbeat on d 33. Statistical analyses were done with PROC MIXED from SAS Institute Inc. Concentrations of P4 were greater from d 8 onward in the hCG group. Concentrations of PAG did not differ between groups from d 17 to 33, suggesting no effect of increased P4 on hastening embryonic attachment and placental development. Nevertheless, PAG was greater in the hCG group on d 47 and 61, suggesting greater placental area or PAG secretory capacity. Pregnancy loss between d 20 and 33 occurred in 24.6% of cows. About 50% of pregnancy loss was due to luteal regression and about 50% was due to conceptus failure; that is, a decrease in PAG in the absence of luteal regression. In conclusion, increased P4 does not hasten embryonic attachment or early placental development but it leads to increased PAG in the second half of the second month of gestation. Additionally, pregnancy loss seems to be initiated by either corpus luteum regression or conceptus failure.

Effects of human chorionic gonadotropin on the last day of a 5-day CIDR Synch protocol and 5 days later on circulating progesterone concentrations and luteal area in Holstein heifers.

The objective was to determine the effect of replacing GnRH with human chorionic gonadotropin (hCG) on the last day of a 5-d CIDR Synch protocol (d 0) and inducing accessory corpus luteum (CL) formation with hCG 5 d later (d 5) on serum progesterone (P4) concentrations and luteal dynamics in dairy heifers. Holstein heifers (n = 207) were synchronized with a 5-d controlled internal drug release (CIDR) Synch protocol (d -8: used CIDR inserted; d -3: CIDR removed and PGF2α). Heifers were randomly assigned to 1 of 4 treatments on d 0: control (G; n = 55), H (n = 50), GH (n = 53), and HH (n = 49). Heifers in G were treated with 100 µg of GnRH on d 0, while H heifers received hCG (3,300 IU) on d 0. Heifers enrolled in GH were treated with GnRH on d 0 and hCG on d 5, while HH received hCG on d 0 and 5. Ovaries were scanned by ultrasound on d 0, 5, and 12, and blood was collected on d 0, 5, 7, and 12. Heifers that ovulated before or after the hCG or GnRH d 0 treatment and had P4 ≤ 0.50 ng/mL on d 0 were considered as synchronized. Overall protocol synchronization response was 93.8%, with no differences among treatments. Only synchronized heifers (n = 193) were included in the analyses of luteal dynamics after d 0. Serum P4 concentration and original CL luteal area on d 5 in heifers treated with hCG on d 0 (H + HH) were greater than in heifers treated with GnRH on d 0 (G + GH). Almost all heifers treated with hCG on d 5 had ≥2 CL on d 12 (98.6%). Ovulatory response for d 5 hCG treatment did not differ for GH versus HH (97.2 vs. 94.7%). Heifers in HH had the highest serum P4 on d 7, and G had the lowest serum P4 on d 7 and 12. In contrast, serum P4 on d 7 did not differ for H versus GH. On d 12, serum P4 and total luteal area were not different for GH versus HH. In summary, heifers that received hCG on d 0 had a larger total luteal area and greater serum P4 concentration on d 5 than heifers treated with GnRH on d 0. Moreover, hCG on d 5 promoted a greater proportion of heifers with ≥2 CL on d 12 and a larger luteal area of the original CL, which resulted in a larger total luteal area on d 12. The HH treatment successfully increased serum P4 concentrations in heifers on d 7 compared with the other treatments.

Accessory corpus luteum induced by human chorionic gonadotropin on day 7 or days 7 and 13 of the estrous cycle affected follicular and luteal dynamics and luteolysis in lactating Holstein cows.

Our objective was to determine the effect of inducing an accessory corpus luteum (CL) with human chorionic gonadotropin (hCG; 3,300 IU) on d 7 (hCG7) or 2 accessory CL with hCG on d 7 and 13 (hCG7+13) of the estrous cycle in noninseminated lactating Holstein cows. Cows (n = 86) between 39 and 64 DIM were pretreated with an Ovsynch + CIDR protocol, and only synchronized cows were used (n = 64). The day of the last GnRH of Ovsynch was considered d 0 of the estrous cycle. Follicular and luteal dynamics of cows were evaluated daily during an entire estrous cycle by ovarian ultrasonography. Blood samples were collected daily to measure serum concentration of progesterone (P4). Cows were randomly assigned to CON (n = 22, no treatment), hCG7 (n = 20), or hCG7+13 (n = 22) treatments. Two cows from hCG7+13 failed to ovulate after hCG and were removed from the analyses post-hCG treatment. The first day of luteolysis was considered the day that P4 declined to more than 2 SD of the mean for the 4 consecutive P4 concentrations with the greatest mean in late diestrus for each individual cow. The P4 cut-off for complete luteolysis was <1.0 ng/mL. Mean P4 on d 7 (3.23 ± 0.16 ng/mL) did not differ among treatments. Cows treated with hCG had greater total luteal and original CL volume and serum P4 during diestrus than CON. Cows treated with hCG7+13 had greater serum P4 after d 13 of the cycle than hCG7. Cycles were classified as having atypical cycles if the dominant follicle or future dominant follicle at the time of luteolysis did not ovulate (delayed ovulation; CON, n = 2; hCG7, n = 4; hCG7+13, n = 3), had a short cycle (CON, n = 1), delayed (CON, n = 2) or incomplete luteolysis (CON, n = 1; hCG7, n = 4; hCG7+13, n = 5). The remainder of cycles with normal complete luteolysis followed by ovulation were considered to be typical. Based on blood perfusion, the CON cow with incomplete luteolysis had 2 original CL remaining functional after first onset of luteolysis. The rest of the cows with incomplete luteolysis (9/10) had one or more CL regressing and at least one remaining functional after first onset of luteolysis. No specific pattern for CL side (ipsilateral vs. contralateral to a CL with complete regression) was observed for nonregressed CL. Cows with incomplete luteolysis had a second onset of luteolysis to undergo complete functional luteolysis. The proportion of cows with typical cycle was 73% (16/22) for CON, 60% (12/20) for hCG7, and 55% (11/20) for hCG7+13. Cows with typical cycles treated with hCG (hCG7 and hCG7+13) had a later onset of luteolysis, prolonged time to undergo complete luteolysis, and greater proportion of cows with 3 follicular waves than CON, resulting in a longer interovulatory interval for hCG7 and hCG7+13 than CON. In summary, accessory CL induced by hCG during diestrus not only altered follicular and luteal dynamics but also deferred and prolonged the luteolytic process.

Effect of PGF2α treatments during early corpus luteum development on circulating progesterone concentrations and ovulation in breeding-age Holstein heifers.

The objective of this study was to test the effect of low circulating concentrations of progesterone (P4) on pre-ovulatory follicle development in heifers as part of an overarching objective to develop a model to understand this phenomenon in dairy cattle without the confounding factors of lactation. Holstein heifers between 12 and 13 mo of age were pre-synchronized to ensure all heifers were on d 6 of the estrous cycle at the start of the Ovsynch program. Only heifers with CL regression and ovulation to the following pre-treatment strategy were used in the study: 0.5 mg cloprostenol (PGF2α), 2 d later, 0.1 mg GnRH, 6 d later, GnRH (G1; 1st GnRH of Ovsynch). Heifers (n = 159) responding to pre-treatment were randomly assigned to 4 groups and completed the Ovsynch program: high P4 control (HPC), low P4 control (LPC; PGF2α 24 h after G1), PG2 (PGF2α 24 and 48 h after G1) and PG3 (PGF2α 24, 48, and 96 h after G1). Only heifers that had ovulation to G1 remained in the study. Blood samples were collected in all heifers on d 7 (n = 157) and in a subset of heifers on d 1, 2, 3, 4 (n = 82) after G1 to measure serum P4. Pre-ovulatory follicle size at G1 (13.0 ± 0.1 mm; P = 0.53) and mean serum P4 24 h after G1 (d 1; 3.62 ± 0.11 ng/mL; P = 0.46) did not differ among treatments. HPC heifers had greater (P < 0.001) mean serum P4 compared to LPC, PG2 and PG3 on d 2, 3, 4, and 7. On d 2, 3 and 4, mean serum P4 of LPC, PG2 and PG3 heifers did not differ (P > 0.10). On d 7, LPC heifers had greater (P < 0.001) serum P4 compared to PG2 and PG3 heifers. Mean ± SEM serum P4 on d 7 after G1 was 8.43 ± 0.39, 2.55 ± 0.36, 1.58 ± 0.20, and 1.21 ± 0.15 ng/mL for HPC, LPC, PG2, and PG3, respectively. Percentage of heifers with P4 < 0.50 ng/mL on d 7 was greater (P < 0.05) for LPC, PG2 and PG3 (27, 32 and 26%, respectively) compared to HPC (0%). A greater (P < 0.05) proportion of heifers ovulated before G2 in the LPC, PG2 and PG3 than in the HPC. For heifers that ovulated after G2, low serum concentrations of P4 in LPC, PG2 and PG3 induced double ovulations in 6/97 heifers after the final GnRH of Ovsynch compared to 0/33 in HPC. In summary, PGF2α treatments during early CL development reduced circulating P4 concentrations 7 d after G1 compared with both HPC and LPC. However, it did not effectively control CL and follicle function to be utilized as a model to test high vs. low serum P4 on fertility parameters in Holstein heifers.

Effects of GnRH and hCG administration during early luteal phase on estrous cycle length, expression of estrus and fertility in lactating dairy cows.

Our objective was to determine the effects of a single treatment of human chorionic gonadotropin (hCG) or GnRH from d 5 to 7 of the estrous cycle on cycle length, expression of estrus and fertility in lactating dairy cows. Lactating Holstein cows (n = 354) located in Farm 1 and lactating Jersey cows located in Farm 2 (n = 210) detected in estrus by an Automated Activity Monitor (AAM) system from 27 to 50 days in milk (DIM) were randomly assigned to receive one of three treatments from d 5 to 7 of the estrous cycle: control (untreated; CON; Holstein, n = 111; Jersey, n = 66), GnRH (86 µg gonadorelin acetate im; Holstein, n = 116; Jersey, n = 75), or hCG (3,300 IU im; Holstein, n = 127; Jersey, n = 69). Ovaries were scanned with ultrasound in a random subgroup of cows (Holstein/Farm 1, n = 147; Jersey/Farm 2, n = 94) on the day of treatment and 3 or 4 d later to determine ovulation. Estrus was detected after treatment by an AAM, and peak activity and heat index were recorded. A random subgroup of cows observed in estrus after treatment received first AI from 51 to 80 DIM (Holstein, n = 208; Jersey, n = 138). Pregnancy diagnoses were performed by transrectal ultrasonography at 37 ± 3 d post-AI. Holstein and Jersey cows treated with GnRH and hCG had an increased (P < 0.05) ovulatory response compared with controls. Human chorionic gonadotropin decreased (74%; P = 0.05) and GnRH tended to reduce (75%; P = 0.07) the proportion of multiparous Holstein cows returning to estrus compared with CON (86%). Cows treated with hCG had a longer (P < 0.01) estrous cycle length (24.6 ± 0.3 d, Holstein; 23.0 ± 0.3 d, Jersey) compared with CON cows (22.7 ± 0.3 d, Holstein; 21.3 ± 0.3 d Jersey) and GnRH (22.9 ± 0.3 d, Holstein; 21.1 ± 0.3 d Jersey). The percentage of cows with high (≥80) peak activity and heat index did not differ (P > 0.50) between treatments, and milk production did not affect (P > 0.65) the duration of estrus. Pregnancy per AI (P/AI) was not affected by treatments in Holstein (P = 0.93; CON: 34.3%, GnRH: 35.4%, and hCG: 31.5%) and in multiparous Jersey cows (P = 0.35; CON: 34.3%, GnRH: 35.4%, and hCG: 31.5%), but hCG had greater (P = 0.03; 55%) P/AI than GnRH (30.0%) and a trend (P = 0.06) for greater P/AI than CON (33.3%) in primiparous Jersey cows. In summary, inducing the formation of an accessory corpus luteum from d 5 to 7 of the estrous cycle with hCG reduced expression of estrus in multiparous Holstein cows. Moreover, hCG increased estrous cycle length in Holstein and Jersey cows, and it did not affect first service P/AI at 37 ± 3 d post-AI in Holstein and multiparous Jersey lactating cows. However, hCG increased P/AI in primiparous Jersey cows. Future research with a larger number of cows is needed to confirm these intriguing fertility results.

Presynchronization of lactating dairy cows with PGF2α and GnRH simultaneously, 7 days before Ovsynch have similar outcomes compared to G6G.

The overarching objective of this study was to develop an alternative strategy for first and greater services that will improve fertility in lactating dairy cows for dairy operations limited by labor or other logistical constraints that make it difficult to use Presynch-11, G6G, or Double-Ovsynch. Our overall hypothesis was that simplification of a Presynch program through the combination of PGF2α and GnRH on the same day (PG + G), 7 days before the first GnRH of Ovsynch, would allow for similar ovulation and luteolysis rate and pregnancies per AI (P/AI) compared with G6G. Lactating dairy cows 58 to 64 days in milk (first service; n = 114), and cows diagnosed not pregnant 39 days after previous AI (second + service; n = 122) were blocked by parity and service and randomly assigned to control or PG + G. Control cows received G6G (n = 116) that consisted of PGF2α, 2-day GnRH, 6-day GnRH, 7-day PGF2α, 56-hour GnRH, and 16-hour AI. Treated cows (PG + G; n = 121) received PGF2α and GnRH, 7-day GnRH, 7-day PGF2α, 56-hour GnRH, and 16-hour AI. All cows received a second PGF2α 24 hours after the PGF2α of Ovsynch. First service cows received AI between 76 and 82 days in milk and second + service received AI 57 days after previous AI. Pregnancies/AI (n = 230) were similar in controls compared with treated cows on Day 35 (57 vs. 50%; P = 0.27) and Day 49 (54 vs. 47%; P = 0.33), respectively. Percent of cows ovulating after GnRH of the presynchronization was greater (P = 0.002) for controls vs. treated (80 vs. 58%); however, ovulation after first GnRH of Ovsynch was similar (67 vs. 68%; P = 0.86). Serum concentrations of progesterone were similar (P = 0.78) at the time of first GnRH of Ovsynch for control and treated cows (2.22 vs. 2.14 ng/mL). However, serum progesterone at the time of PGF2α of Ovsynch was greater (P = 0.002) for control cows compared with treated cows (5.75 vs. 4.64 ng/mL). In summary, administering both PGF2α and GnRH on the same day, 7 days before the start of Ovsynch, appears to be a simple alternative that results in acceptable P/AI but potentially less progesterone during the growth of the ovulatory follicle.

Impact of circulating concentrations of progesterone and antral age of the ovulatory follicle on fertility of high-producing lactating dairy cows.

Dairy cow infertility negatively affects profit of dairy production enterprises around the world, and enhancing conception rates of dairy cows is a critical management issue to resolve. It appears that conception rates of dairy cows are attenuated due to reduced progesterone concentrations in circulation during growth of the ovulatory follicle. It is not clear how reduced progesterone influences fertility, but data presented in this brief review suggest that it can be somewhat reversed through increasing concentrations of progesterone during the growth of the ovulatory follicle before luteolysis. Ovsynch protocols may be utilised to enhance progesterone concentrations through the induction of an accessory corpus luteum (CL) following the initial gonadotropin-releasing hormone (GnRH) treatment. Cows at Day 13 of the oestrous cycle with a 7-day-old accessory CL had ~50% more progesterone at the time of prostaglandin injection of Ovsynch compared with cows with only a Day 13 CL. Ovsynch can consistently induce an accessory CL following the initial GnRH treatment if cows are on Days 6 or 7 of the oestrous cycle at the time of treatment. Pre-synchrony strategies are critical to enhance the probability that cows will be on Days 6 or 7 at first GnRH treatement of Ovsynch.