Actores Institucionales

Herd Management

Experiments 1 and 3 were conducted at the Kansas State University Dairy

Teaching and Research Center, Manhattan. Experiment 2 was conducted at Kansas State University as well as at 3 commercial northeast Kansas locations. All research at Kansas State University was conducted from October 2005 until October 2006. Research at the 3 commercial locations was performed between March and November 2006. Cows were housed in covered freestalls bedded with sand at all locations. All pens were covered and water was applied by sprinklers during summer months. Fans over freestalls, feed lines, or both were also in place at the 3 commercial locations. All cows were fed twice or thrice daily a TMR that met or exceeded National Research Council (NRC, 2001) requirements for lactating cows. Diets consisted primarily of chopped alfalfa hay, wet corn gluten meal, corn silage, whole cottonseed, soybean meal, and corn grain, plus a vitamin-mineral premix. Cows had ad libitum access to fresh water. Table 1 summarizes other herd management information.

Experimental Approach

Experimental approach is presented in Figure 1. Seven d before pregnancy diagnosis, dairy cows, along with a few nulliparous dairy heifers (herd 1 only), were assigned randomly to treatments of hCG, GnRH, or saline. Treatments were assigned based on lactation number, number of previous AI, and last test-day milk yield (cows only). Pregnancy was diagnosed 1 wk later (d 0).

Experiment 1

Ovaries of Holstein cows and heifers in herd 1 were examined by transrectal ultrasonography (5.0 MHz linear-array transducer, Aloka 500V; Corometrics Medical Systems, Inc., Wallingford, CT) and structures were mapped, sized, and recorded. Cattle received a treatment of saline, GnRH (100 µg; 2 mL of Fertagyl, Intervet Inc., Millsboro, NJ), or either of 4 doses (500, 1,000, 2,000, or 3,000 IU) of hCG (0.5, 1, 2, or 3 mL of Chorulon, Intervet Inc.). Cows were then re-examined 1 wk later and those follicles that were induced to ovulate were noted.

All variables (ovulation incidence, number of females having at least 1 follicle ≥ 8 mm in diameter, number of follicles ≥ 8 mm, follicles ≥ 8 mm that ovulated and pregnancy rate) were analyzed by using ANOVA (procedure GLM; SAS Inst. Inc., Cary, NC) in a model that included treatment (n = 6), stage (d 22 to 28 or d 29 to 35 post-AI) at treatment injection, and the interaction of treatment × stage. A priori contrast was constructed to test all hCG doses ≥ 1,000 IU vs. other treatments.

Experiment 2

One wk before pregnancy diagnosis, dairy cows at 4 Kansas locations were assigned to receive 100 µg of GnRH (Fertagyl, Intervet Inc.), 1,000 IU of hCG

(Chorulon, Intervet Inc.), or left as untreated controls based on lactation number, number of previous AI, and last test-day milk weight. Cows were diagnosed for pregnancy by transrectal ultrasonography on d 30 to 43 (herd 1) or by transrectal palpation on d 37 to 45 (herds 2 to 4) post-insemination. The same veterinary practitioner diagnosed pregnancy in herds 2, 3, and 4. When cows (n = 1,235) were diagnosed pregnant, the resynchronization protocol was discontinued and pregnancy status was reassessed 4 to 9

wk later in herds 2, 3, and 4 and 9 wk later in herd 1 to determine pregnancy survival. Cows diagnosed not pregnant (n = 1,748) were given PGFB2α Bat diagnosis and received one TAI 72 h later. Cows at 3 locations were administered 100 µg of GnRH (Fertagyl,

Intervet Inc.) at the time of AI, whereas cows in herd 1 were given GnRH 16 to 24 h before TAI. Following TAI, all cows not detected in estrus and inseminated were again diagnosed for pregnancy 30 to 45 d later. Some nonpregnant cows in the 3 commercial dairies were inseminated early based on activity, standing estrus, and chalk rubs. These cows were eliminated from the results and were not included in analyses.

Palpation pregnancy rate was calculated as the number of pregnant cows at each diagnosis divided by the number of cows presented for pregnancy diagnosis. Pregnancy rate was calculated as the number of pregnant cows at each diagnosis divided by the number of cows previously inseminated and treated. Pregnancy survival between the first and second pregnancy diagnosis (4 to 9 wk later) also was determined. Pregnancy rates and pregnancy survival were analyzed by using ANOVA (procedure GLM; SAS Inst. Inc.). The model for pregnancy survival consisted of treatment (n = 3), lactation number (1, 2, or ≥3), herd (n = 4), interaction of treatment × lactation number, interaction of treatment × herd, season of treatment nested within herd, and most recent test-day milk weight (covariable). A priori contrasts were constructed for each treatment vs. control. The model for pregnancy rate also included sire and technician, each nested within herd.

Experiment 3

At 1 location (herd 1), transrectal ultrasonography was conducted at the initiation of the resynchronization protocol before treatment. Ovarian structures were mapped and follicles were sized. Follicular diameter was determined by averaging the largest cross-

sectional width and height measured by ultrasound electronic calipers. Structures were monitored again 7 d later and new corpora lutea (CL) which were not present or visible at the first ultrasound exam were noted. New CL corresponding to large follicles at the first ultrasound were assumed to have ovulated in response to saline, 100 µg of GnRH, or 1,000 IU of hCG.

Blood samples were collected from a coccygeal blood vessel at the time of each ultrasonography exam. Samples were stored on ice until transported to the laboratory for centrifugation 24 h later. The serum portion was retained, frozen, and serum

concentrations of progesterone were later quantified by RIA (Skaggs et al., 1986). Intra- and interassay coefficients of variation for 11 assays were 8.9 and 9.5%, respectively, for a pooled sample that averaged 3.85 ± 0.1 ng/mL.

Incidence of ovulation was analyzed by using ANOVA (procedure GLM; SAS Inst. Inc.) with a model consisting of treatment (n = 3), lactation number (1, 2, or ≥3), pregnancy status, stage of follicular development (d 22 to 28 or d 29 to 35 post-AI), follicle class (≤10, 11-12, 13-14, or >14 mm), number of CL before treatment (0, 1, or ≥2), number of follicles ≥ 8 mm at treatment (0, 1, 2, or 3), breeding cluster (n = 28), interaction of treatment × lactation, treatment × pregnancy, treatment × stage, and treatment × beginning number of CL. A priori contrasts were constructed for the combined treatments vs. control and GnRH vs. hCG. Incidence of ovulation based on probable days of the cycle at treatment also was analyzed using ANOVA (procedure GLM; SAS Inst. Inc.) with a model similar to that above. This reduced model condensed stage to 2 groups, each consisting of 3 d (d 23 to 25 and d 31 to 33 post-AI at treatment

injection) in which the majority of cows had a follicle with a high probability of ovulating.

Concentrations of progesterone from 7 d after treatment also were analyzed by using ANOVA (procedure GLM; SAS Inst. Inc.). The model was similar to the one used to analyze incidence of ovulation, but did not include follicle class, beginning number of CL, or number of follicles ≥ 8mm. The model included however, the ending number of CL (original + induced CL) and the interaction of treatment × ending number of CL, plus the concentration of progesterone at pregnancy diagnosis as a covariable adjustment. A priori contrasts were constructed for both treatments vs. control and GnRH vs. hCG.

RESULTS

Experiment 1

Results of this experiment are summarized in Table 2. More than 95% of the females had at least 1 follicle ≥ 8 mm in diameter. Average number of follicles at least 8 mm or more in diameter per female ranged from 1.4 ± 0.2 to 1.9 ± 0.2 in each group of females before treatment. Ovulatory responses (shown as the percentage of cows with a new CL in Table 2) per female treated with saline, GnRH, and 500 IU of hCG were exceeded (P < 0.05) by the larger doses (1,000 IU or greater) of hCG. When the combined hCG doses ≥ 1,000 IU were compared with only controls, the P value was 0.06. When ovulatory response was calculated based on the total numbers of follicles, percentage responses were similar to those on a per-female basis. Compared with , control, GnRH, and 500 IU of hCG, the greater doses of hCG produced more (P < 0.05) ovulations. A tendency (P = 0.12) occurred for more follicles to ovulate after at least 1,000 IU of hCG than after saline alone.

Experiment 2

Herd palpation pregnancy rates ranged from 33.5 to 39.6% in the 4 herds (Table 3). Herd 4 had greater palpation pregnancy rates than herds 2 (P = 0.04) and 3 (P = 0.003). Pregnancy survival 4 to 9 wk after initial pregnancy diagnosis by treatment based on postpartum insemination number is illustrated in Table 4 for 1,236 cows. Overall, no difference in pregnancy survival was detected between cows treated with hCG (93.6%; n = 420) 7 d before pregnancy diagnosis and those left as untreated controls (95.3%; n = 403). Pregnancy survival, however, tended (P = 0.06) to be reduced in those cows treated with GnRH (93.0%; n = 413) compared with controls. Herd (P = 0.004) and season (P < 0.05) affected pregnancy survival. Herd tended to have or had an effect on pregnancy survival at the first (P = 0.10), second (P < 0.01), and third (P = 0.002) inseminations post-AI. Herds 1, 2, 3, and 4 had survival rates of 85.1, 99.6, 91.2, and 94.2%, respectively. Neither lactation number nor last test-day milk weight had an effect on pregnancy survival. Lactation number, however, tended (P = 0.11) to affect

pregnancy survival for cows that conceived at their first postpartum insemination, with older cows having less survival (89%) than first-lactation cows (95%).

A treatment × herd interaction (P = 0.004) is illustrated in Figure 2. In herd 1, cows treated with hCG exhibited the greatest pregnancy survival; whereas survival was compromised in cows treated with GnRH compared with controls. Herd 3, however, exhibited reduced survival in females treated with hCG compared with those treated with GnRH and those left as untreated controls. Herds 2 and 4 responded similarly to

treatment with survival rates being comparable across all treatments.

significant difference (P = 0.17) in pregnancy rate was detected between cows treated with hCG (16.5%; n = 541) and those treated with GnRH (17.9%; n = 703) or left as untreated controls (12.9%; n = 505) 7 d before pregnancy diagnosis. Pregnancy rate for GnRH- treated cows, however, tended (P = 0.08) to be greater than that of controls. Treatment, herd, lactation, nor treatment × lactation interaction had any effect on the risk of pregnancy. In contrast, for each 10 kg increase in last test-day milk weight, pregnancy rate decreased (P < 0.05) by 2.2 ± 1%. Sire nested within herd (P = 0.007) and season nested within herd (P = 0.018) had an effect on pregnancy rate, whereas technician nested within herd did not (P = 0.79).

Figure 3 illustrates a treatment × herd interaction (P < 0.05) on pregnancy rate. Herds 1 and 4 responded similarly to treatment as did herds 2 and 3. Cows treated with hCG and those treated with GnRH had the greatest pregnancy rates numerically in 2 herds each. Untreated controls seemed to have reduced pregnancy rates in 1 herd, but fertility was comparable with either GnRH or hCG in the other 3 herds.

Experiment 3

Ovaries of 490 cows were monitored for ovulation 7 d after treatment (pregnancy diagnosis) with hCG, GnRH, or saline. Treatment affected (P < 0.001) incidence of ovulation with 52.4% (n = 185), 39.1% (n = 151), and 20.1% (n = 154) of hCG, GnRH, and control cows ovulating, respectively. Treatment with hCG did not result in more (P = 0.20) cows ovulating than treatment with GnRH. Treatment with hCG or GnRH resulted in more (P < 0.001) cows ovulating than treatment with saline. Percentage of cows having at least 1 new CL by 7 d after treatment is summarized in Figure 4. Among nonpregnant cows, no difference was detected between hCG and GnRH treatments in the

appearance of new CL. Treatment with hCG (P = 0.07) tended to produce more

accessory CL in pregnant cows as indicated by a treatment × pregnancy status interaction (Figure 4).

Number of CL already present at the time of treatment affected (P < 0.001) the incidence of ovulation. Cows with no CL present (n = 98) ovulated 62.2% of the time, whereas cows with 1 (n = 261) and ≥ 2 (n =112) CL ovulated 36.0 and 24.1% of the time, respectively. A tendency (P = 0.08) for a treatment × beginning number of CL

interaction also was detected (Figure 5). Interestingly, GnRH produced a greater incidence of ovulation when no spontaneous CL was present at the time of treatment.

Neither lactation number, pregnancy status, nor treatment × lactation number influenced incidence of ovulation. Diameter of the follicles present at the time of treatment had no significant effect on the incidence of ovulation. Also not significant was the putative follicular wave as described for each cow at treatment based on days since timed AI (Table 6).

Control cows ovulated fewer (P < 0.05) follicles than those cows treated with GnRH or hCG during the first follicular wave (d 23 to 25; Table 7). Proportion of cows that ovulated more than 1 follicle in response to treatment was 24.6% (n = 69) when treatments occurred at d 29 to 35 compared with 16.5% (n = 121) at d 22 to 28. Average CL per cow was greater (P < 0.05) for the latter treatment period (1.3 ± 0.08 vs. 1.1 ± 0.06) for cows that ovulated.

Blood samples were collected from 486 cows at the time of treatment 7 d before pregnancy diagnosis and again at pregnancy diagnosis (d 0). Table 8 summarizes blood serum concentrations of progesterone 7 d after treatment, based on treatment, pregnancy

status, and number of CL. Concentrations of progesterone 7 d after treatment were adjusted for concentrations of progesterone before treatment. Treatment had no effect on concentrations of progesterone. As expected, pregnant cows (n = 166) had greater (P < 0.001) concentrations of progesterone than nonpregnant cows (n = 320). Also not surprising is the observation that concentrations of progesterone increased as the number of CL present at collection of the second blood sample increased from 0 to ≥ 2 CL. Stage post-AI (n = 2) did not affect concentrations of progesterone for cows in which

treatments were initiated at d 22 to 28 or d 29 to 35.

DISCUSSION

Experiment 1

Many researchers (Wiltbank et al., 1961; de los Santos-Valadez et al., 1982; McDermott et al., 1986; Price and Webb, 1989; Howard and Britt, 1990; Howard et al., 1990; Stevenson et al., 2007) have used hCG to induce ovulation in dairy and beef cattle. Our study, however, was first to determine the minimum effective dose needed to induce ovulation in cattle. A distinct increase in number of females that ovulated and percentage of total follicles that ovulated is evident in those cows treated with ≥ 1,000 IU of hCG. No advantage, however, was detected for the larger doses (2,000 or 3,000 IU) of hCG.

Some ovulations in each treatment were spontaneous due to the stage of cycle at treatment and some CL were immature at the time of first observation and were not visible until the second examination occurred 7 days later. These occurrences are

accounted for by detected ovulations after saline treatment and provide some evidence as to what percentages of ovulations were actually induced by treatment with GnRH or

hCG. From Exp. 1, we concluded that a dose of 1,000 IU of hCG exceeded the ovulatory induction of saline, GnRH, and the smallest dose of hCG (500 IU) in these dairy females.

Experiment 2

Pregnancy survival tended to be reduced in cows treated 7 d before pregnancy diagnosis with GnRH compared with untreated controls. This reduction is similar to another report in which pregnancy survival was reduced when third or greater lactation cows were treated with GnRH, but at 4 to 9 d after insemination (Stevenson et al., 2007). Because treatment with GnRH did not increase concentrations of progesterone over controls, no change in pregnancy survival should be expected. The mechanism behind a possible increase in pregnancy mortality, however, is unknown. Careful interpretation of this reduction in pregnancy survival by GnRH should be noted because it was clearly evident only in 1 herd, although in 2 of the remaining herds, pregnancy survival was reduced by 0 to 3.5% units (Figure 2). Pregnancy survival in hCG treated cows did not differ statistically from GnRH treated cows or controls. This is surprising because the results from Exp. 3 revealed that more accessory CL were formed after hCG in pregnant cows than after GnRH or saline. Perhaps accessory CL formation was too late (d 23 to 38) to benefit pregnancy survival. As a result of differences in genetics and management practices among herds, a herd effect on pregnancy survival is not surprising. Cavestany et al. (1985) demonstrated the effects of heat stress on fertility in Holstein cattle. Because the experiment encompassed all seasons, the seasonal effect on pregnancy survival also is to be expected.

Pregnancy rate tended (P = 0.08) to be greater in cows initiating the resynchronization protocol with GnRH than in untreated controls. The logical

explanation for this is simply that more follicles ovulated in response to GnRH, and thus, more cows had a synchronized follicle at the time of AI. By this logic, however,

pregnancy rate in hCG treated cows also should be greater than that of controls because more follicles were induced to ovulate after hCG than in controls. Howard and Britt (1990) discovered that an hCG-induced CL behaves differently than a spontaneously induced CL when they observed that the former was induced by PGFB2αB to regress before d 5 of its existence. Fricke et al. (1993) also observed that hCG-induced CL differed in size and color from spontaneously formed CL and produced less progesterone when cultured in vitro in the presence of LH. It is a reasonable assumption that physiological differences in hCG-induced CL could hinder pregnancy rates at a later insemination.

As with pregnancy survival, a treatment × herd interaction was detected for

pregnancy rate. Because the pregnancy diagnosis schedule of herd 1 differed from that of the other 3 herds, one might expect the first herd to respond differently to treatment than the others. This, however, is not the case because pregnancy rates in herds 1 and 4 were similar and those of herds 2 and 3 were similar (Figure 3).

We conclude that there is no distinct advantage to hCG over GnRH, or vice versa, in pregnancy survival or pregnancy rate in this resynchronization protocol. Cows in 3 of 4 herds exhibited increased pregnancy rates when Resynch-Ovsynch was initiated with hCG. Economically, a 100 µg dose of GnRH is similar in cost to 1,000 IU of hCG. We conclude, however, that from a dairy producer viewpoint the evidence does not merit a change from GnRH to hCG. Even though untreated controls exhibited the numerically greatest incidence of pregnancy survival, it is not a feasible option to initiate the Resynch-Ovsynch protocol with saline because pregnancy rates in those females

subsequently diagnosed not pregnant and resubmitted for insemination were reduced by more than 38% compared with GnRH.

Experiment 3

The treatment × pregnancy status interaction was a result of many more accessory CL formed in pregnant cows treated with hCG than with GnRH. This is probably a function of hCG’s increased half-life compared with GnRH-induced LH release in response to hCG’s 4 sites of O-linked glycosylation (Jameson and Hollenberg, 1993). For example, hCG bound to the LH receptor in luteal cells is internalized 50 times slower than LH (Niswender et al., 1985). This allows hCG to be more potent when inducing ovulation. A portion of the new CL detected in nonpregnant control cattle is probably a result of extended estrous cycles (> 22 d). Spontaneous CL in these cattle would have been undetectable at the time of first ultrasound diagnosis. Some of these CL also were detected because cycles in cows were not synchronized at the initial AI and ovulated spontaneously between scans. Seven percent of pregnant cows left as untreated controls ovulated between ultrasound examinations. It is not known what percentage of cows ovulate spontaneously after pregnancy is initiated. A portion of this 7% likely resulted from technician error.

A novel finding of this study was that in cows with no spontaneous CL present at the time of treatment, GnRH induced more follicles to ovulate than hCG. A similar number of ovulations were observed between hCG treated cows and controls when no CL were present at the time of treatment. When 1 or more CL were already present,

however, hCG induced more ovulations than GnRH or saline. When all treatments are pooled, the incidence of ovulation decreased as the beginning number of CL increased.

Table 6 provides an in depth look at the incidence of ovulation based on days since AI at treatment. Days 23 to 25 and 31 to 33 were considered to be the probable first and second follicular waves since the first eligible estrus after AI in nonpregnant cows. Interestingly, follicular wave dynamics also can be observed in pregnant cows as well. In first-wave cattle, the incidence of ovulation was reduced in control cows compared with cows receiving treatment. Once again, reasons for ovulation in these cows are varied and include: lengthened estrous cycles, previously unsynchronized cycle at AI, and technician error. When only cattle that fall into the expected wave categories are considered, or in