Complete Luteolysis and Pregnancy Per Insemination After Complete Luteolysis and Pregnancy Per Insemination After Modifying the Standard 7-day Ovsynch Program in Dairy Cows Modifying the Standard 7-day Ovsynch Program in Dairy Cows

Summary Two experiments were conducted with Holstein-Friesian cows in the Republic of North Macedonia and with Holstein cows in Kansas. We hypothesized that a single standard dose of PGF 2α injected on day 8 instead of day 7 in the standard Ovsynch program would increase the proportion of cows with complete luteolysis and increase the synchronization risk compared with control cows receiving a standard dose on day 7. Cows were treated with the standard program (Ov-7x1) or with Ov-8x1 experimental program in experiment 1, using only a single dose of PGF 2α . In experiment 2, a third treatment was added (Ov-7x2), in which a second dose of PGF 2α was administered 24 hours after the first dose. Overall, the results in experiment 1 demonstrated a greater percentage of multiparous cows in the OV-8x1 treatment had complete luteal regression compared with multiparous Ov-7x1 cows, whereas treatments were equally effective in primiparous cows. In contrast, results in experiment 2 revealed nearly 100% of cows in the Ov-7x2 treatment receiving the second dose of PGF 2α had complete luteol-ysis. In both experiments, when the status of luteal function before PGF treatment was examined, the treatments were equally effective in causing complete luteal regression. Pregnancy rates, however, did not differ among treatments indicating that any of the three treatments will likely produce similar pregnancy outcomes with the flexibility of applying either the 7-vs. 8-day treatments.


Introduction
Fixed-time insemination programs have been adopted by dairy managers to facilitate 100% artificial insemination submission rate and reduce the dependence on the detection of estrus. Adoption of various on-farm reproductive technologies increased rapidly in just 7 years based on surveys conducted in 2006 and repeated in 2013 in 17 of the major U.S. dairy states representing 80% of the dairy operations and more than 80% of the dairy cows (National Health Monitoring Survey, Animal Plant Health Inspection Service, USDA). More than 12% of dairy operations used a timed AI program at first services and more than 21% used such programs to resynchronize ovulation in cows diagnosed not pregnant. The original 7-day Ovsynch program (GnRH-1 [day 0] -7 days -PGF 2α -56 hours -GnRH-2 -16 hours -AI) has been modified by further research.

Dairy Research 2020
Maximal pregnancy rate achieved with the Ovsynch program has at least 3 limiting factors: (1) failure of GnRH-1 to induce ovulation of the dominant follicle to initiate a new follicular wave; (2) complete luteolysis after a single dose of PGF 2α ; and (3) failure to induce ovulation after G-2. The proportion of cows ovulating after GnRH-1 was 64% (range of 23 to 96%) and varied by stage of the estrous cycle. Ovulation after GnRH-1 is maximized when the Ovsynch program is initiated between days 5 and 12 of the estrous cycle and this is accomplished by various presynchronization programs (Presynch, PG-3-G, Double Ovsynch, and G-6-G).
Cows exposed to presynchronization and that also ovulated after GnRH-1 generally have a greater pregnancy rate than cows that did not ovulate. Although it is assumed that the latter responses are associated with increased circulating progesterone from the newly formed corpus luteum (CL), during growth of the preovulatory follicle, the progesterone concentration at PGF 2α may not differ between cows with 1 older CL compared with cows with an older and new CL (2 CL). Nevertheless, cows that ovulated after GnRH-1 and formed a new CL are more likely to have an incomplete luteolysis than cows that did not ovulate because a CL less than 7 days old is resistant to regression after a single standard dose of PGF 2α .
Attempts have been made to prevent incomplete CL regression by either increasing the dose of PGF 2α or the frequency of PGF 2α treatments. Increasing the dose of PGF 2α (cloprostenol) from 500 to 750 µg for cows submitted to a 7-day Ovsynch protocol has increased the luteolytic risk, but this effect was only observed in multiparous cows. Inclusion of a second PGF 2α treatment 24 h after the first PGF 2α (days 7 and 8 after GnRH-1) increased the percentage of cows with complete luteolysis from 80 to 97%. Furthermore, a recent meta-analysis demonstrated a clear benefit of an additional PGF 2α treatment during the Ovsynch program on luteal regression (+11.6 percentage units) and on pregnancy rate (+4.6 percentage units).
We hypothesized that a single standard dose of PGF 2α injected on day 8 instead of day 7 after GnRH-1 increases the proportion of cows with complete luteolysis and increases the synchronization risk compared with control cows receiving a standard dose on day 7. We included a treatment with both standard doses of PGF 2α as a positive control. Therefore, our objective was to evaluate the effect of a single standard dose of PGF 2α treatment on day 7, day 8, or on both days on the luteolytic and synchronization risks in lactating Holstein cows inseminated at first service after a standard presynchronization program.

Experiment 1
Lactating Holstein-Friesian cows (n = 347) in one commercial herd located in the southeast area of the Republic of North Macedonia were enrolled in the study. Cows were housed in free-stall barns, and fed a total mixed ration (TMR) once daily to meet or exceed requirements for lactating cows producing 30 L of milk per day. Cows were milked twice daily and had free access to water.
Transrectal ultrasonography was conducted at GnRH-1 to map ovarian structures and at PGF 2α to assess ovulation in response to GnRH-1 (Figure 1). Blood samples to assess concentrations of progesterone were collected at PrePG, GnRH-1, PGF 2α , at 72 hours after PGF 2α (at timed AI), and 7 days after timed AI from the coccygeal vein or artery for later measurement of progesterone.
Complete luteolysis was defined to occur when progesterone was ≥ 1 ng/mL before PGF 2α and ≤ 0.3 ng/mL 72 h later. In addition, when examining the effect of luteolysis, progesterone concentrations at PGF 2α were determined based on the luteal status: (1) cows with only a new CL (cows without a CL at GnRH-1 but ovulated after GnRH-1); (2) cows with an older CL (same CL that was detected at G-1); or (3) both a new and old CL.

Experiment 2
We enrolled lactating 454 Holstein cows weekly for 90 weeks at the Kansas State University Dairy Teaching and Research Center, Manhattan, KS. Cows were housed in free stalls with overhead roofs and fed a TMR twice or thrice (summer) daily calculated to meet nutrient requirements for lactating dairy cows producing 110 lb of 3.5% milk. The diet consisted of alfalfa hay, corn silage, triticale silage, soybean meal, whole cottonseed, ground corn grain, corn-gluten feed, vitamins, and minerals. Cows were milked thrice daily.
Transrectal ultrasonography was conducted at GnRH-1 and before the first or only PGF 2α treatment. Ovaries were scanned by transrectal ultrasonography to determine the Dairy Research 2020 number and location of CL and also the number and diameter of all follicles ≥ 10 mm (mapped and recorded) as in experiment 1.
Blood samples were collected before the first or only PGF 2α treatment and on the day of insemination (70 to 72 hours after the first or only PGF 2α treatment). Complete luteolysis was assessed as in experiment 1.

Experiment 1
Mean number of lactations was 1.9 ± 1.1 with 43.8% of cows in their first lactation. Median days in milk (DIM) at AI were 68 d ranging from 52 to 80 d with a mean ± standard deviation (SD) of 67.7 ± 6.3 d. Herd milk production averaged approximately 66 lb per cow per day.
Progesterone concentrations before GnRH-1, PGF 2a , and AI by treatment are in Figure  2A. Delaying luteolysis by 1 day did not increase progesterone concentrations before PGF 2a , and progesterone did not differ between treatments before GnRH-1 or PGF 2a . Body condition affected progesterone concentrations before PGF 2a as cows with BCS ≥ 2.75 had greater (P < 0.01) progesterone than cows with BCS < 2.75 (8.0 ± 0.3 vs. 6.4 ± 0.3 ng/mL), respectively. In contrast, at the time of AI, regardless of parity, concentrations of progesterone did not different for cows receiving the Ov-7x1 treatment (0.65 ± 0.2 ng/mL), whereas progesterone was less (P < 0.05) in multiparous than primiparous cows (0.45 ± 0.2 vs. 0.17 ± 0.2 ng/mL) receiving the Ov-8x1 treatment.
As expected, progesterone concentrations differed depending on CL status before PGF 2a . Cows bearing only a new CL that formed after ovulation in response to GnRH-1 had the smallest concentration and cows with both an older and new CL had the largest concentration resulting in mean progesterone concentrations that differed (P < 0.01) among the three luteal status groups ( Figure 2B).
Our hypothesis was that lengthening the period between GnRH-1 and PGF 2a by 1 day would increase the proportion of cows with complete luteolysis by the time of AI because new CL forming after GnRH-1 would be 24 hours older and more likely to respond to a single dose of PGF 2a . Of the 347 cows assigned to treatment, 87.9% were cyclic by the onset of treatment, leaving 303 cows to test this hypothesis. More (P = 0.02) multiparous than primiparous cows had complete luteolysis and more (P < 0.05) cows receiving the Ov-8x1 treatment had complete luteolysis than cows in the Ov-7x1 treatment (Table 1). An interaction (P = 0.02) of treatment and parity also was detected. Although complete luteolysis did not differ between treatments in primiparous cows, more (P = 0.02) multiparous cows had complete luteolysis when treated with Ov-8x1 than Ov-7x1 treatment (Table 1). Treatments were equally effective in causing complete luteolysis in cows with only a new CL at the time of PGF 2a (87.5%), an older CL (79.0%), or both (77.7%).

Experiment 2
Mean number of lactations was 1.9 ± 1.1 with 44% of cows in their first lactation. Median DIM at AI was 68 d ranging from 52 to 80 d with a mean ± SD of 67.7 ± 6.3 d. Rolling herd average was 31,324 ± 458 lb during the experimental period.
Delaying luteolysis by 1 day did not increase progesterone concentrations before PGF 2a in the Ov-8x1 treatment. In fact, concentrations of progesterone were less (P = 0.06) in the cows treated with Ov-8x1 compared with the other two treatments ( Figure 3A). Furthermore, progesterone concentrations were greater (P < 0.001) in primiparous cows compared with the multiparous cows (5.2 ± 0.2 vs. 4.3 ± 0.2 ng/mL).
As in experiment 1, progesterone concentrations differed depending on CL status before PGF 2a . Cows bearing only new CL had the smallest concentration and cows with both an older and new CL had the largest concentration resulting in mean progesterone concentrations that differed (P < 0.01) among the three luteal status groups ( Figure 3B).
Cows receiving the second dose of PGF 2a had nearly perfect complete luteolysis, which was greater (P < 0.05) than that observed in cows receiving the other two treatments.
Although not different, complete luteolysis was numerically greater in primiparous than multiparous cows. Complete luteolysis did not differ among cows having different CL status: new CL (91.7%), old CL (95.1%), and both new and old CL (93.3%).
Cows not cycling (n = 18) had numerically few pregnancies than cycling cows (n = 429; 27.8 vs. 41.7%), respectively. Neither treatment nor parity affected pregnancy rate (Table 4). Some variation in pregnancy rates occurred during each season with fall and winter (43.9%) tending (P = 0.08) to be greater than pregnancy rate during spring and summer (36.3%).
The present study aimed to determine the efficacy of injecting PGF 2α on day 8 instead of day 7 to produce more complete luteal regression in the standard Ovsynch protocol in lactating dairy cows. Overall, the results in experiment 1 in Holstein-Friesian cows demonstrated a greater percentage of multiparous cows in the OV-8x1 treatment had complete luteal regression compared with Ov-7x1 cows, whereas treatments were equally effective in primiparous cows. In contrast, results in experiment 2 in Holstein cows, revealed nearly 100% of cows in the Ov-7x2 treatment receiving the second dose of PGF 2α 24 hours after the first dose, had complete luteolysis. In both experiments, when the status of luteal function before PGF treatment was examined, the treatments were equally effective in causing complete luteal regression.
The resulting pregnancy rates achieved were the most important outcomes of these experiments. Pregnancy rates did not differ among treatments, indicating that any of the three treatments will likely produce similar outcomes with the flexibility of the 7vs. 8-day treatments.    Cows were classified at the time of PGF 2α treatment injection as having a new CL (formed in response to ovulation after GnRH-1), an older CL existing at GnRH-1 and present at PGF 2α or both a new and older CL.