Combined Effects of Human Chorionic Gonadotropin and Intravaginal Progesterone Device Treatment in the Early Luteal Phase After Artificial Insemination on Conception Rate in Lactating Dairy Cows

Abstract

The combined effects of human chorionic gonadotropin (hCG) treatment on Day 5 (Day 0 = the day of artificial insemination: AI) and intravaginal progesterone device (IVPD) treatment from Day 5 to 19 on the conception rate and plasma sex steroid hormone were examined in lactating dairy cows. In Experiment 1, cows were divided into a non-treatment group (n = 181), untreated; an IVPD group (n = 74), with an IVPD inserted into the vagina from Day 5 to 19; and an hCG + IVPD group (n = 87), with 3000 IU hCG administered intramuscularly on Day 5 and an IVPD inserted into the vagina from Day 5 to 19. The conception rates of the hCG + IVPD group, which underwent ≤3 AIs (52.8%) and >3 AIs (73.3%), and the IVPD group, which underwent >3 AIs (63.0%), were significantly higher than that of the non-treatment group, which underwent ≤3 AIs (34.2%). In Experiment 2, blood samples were collected from the non-treatment group (n = 6), the IVPD group (n = 6), and the hCG + IVPD group (n = 7) on days 5, 8, 12, and 15 from estrus to measure plasma progesterone (P₄) and total estrogen (E) concentrations. The plasma P₄ concentration of the hCG + IVPD group tended to be higher than that of the non-treatment group on Day 15; however, plasma E concentrations were not different among groups. These results suggest that the conception rate was improved by hCG + IVPD treatment regardless of AI number because of higher plasma P₄ concentrations later in the estrous cycle.

1. Introduction

Reproductive performance in lactating dairy cows is important for economic implications in the dairy industry [1]. Achieving a high conception rate after artificial insemination (AI) is one of the most important factors for improving the reproductive performance in lactating dairy cows [2]. To increase the conception rate after AI, human chorionic gonadotropin (hCG) [3,4,5] or an intravaginal progesterone (P₄) device (IVPD) [6,7,8] is used in the early luteal phase to increase plasma P₄ concentrations. In our previous study, IVPD treatment administered 5 to 19 days after AI increased the conception rate in repeat breeding lactating dairy cows, which were inseminated with more than three AIs. In contrast, the IVPD treatment did not increase the conception rate of cows with no more than three AIs [9]. Therefore, an alternative method must be developed for increasing the conception rate of normal lactating dairy cows after AI.

Treatment with an IVPD increases plasma P₄ concentrations immediately after insertion into the vagina [10]; this is followed by a gradual decrease [11]. Plasma P₄ concentrations seven days after IVPD treatment from the early luteal phase does not differ from those in non-treatment conditions [12]; however, hCG treatment during the early luteal phase increases plasma P₄ concentrations three days after treatment in lactating dairy cows [13]. Because of the formation of an accessory CL after hCG treatment, plasma P₄ concentrations are higher in the later luteal phase [14]. From these facts, it is hypothesized that a combination of IVPD treatment and hCG treatment five days after AI could provide an acute increase and maintenance of higher plasma P₄ concentrations through the estrous cycle. In a previous study, IVPD treatment 15–17 days after AI increases the conception rate of cows without a clinical history of retained placenta [15]. Therefore, this sustained elevation in plasma P₄ concentrations could increase conception rates in normal lactating dairy cows. However, the combined effects of hCG and IVPD treatment during the early luteal phase after AI on conception rate have not been fully evaluated in lactating dairy cows.

The objectives of this study were (1) to evaluate the combined effects of hCG treatment at 5 days and IVPD treatment 5 to 19 days after AI (first AI) on the conception rate, detection rate for return to estrus (re-estrus), conception rate at re-estrus (second AI), and cumulative pregnancy rate (first + second AI), and (2) to evaluate the effects of hCG and IVPD treatment on the dynamics of the corpus luteum (CL), dominant follicle (DF), plasma P₄, and total estrogen (E) concentrations during the estrous cycle in lactating dairy cows.

2. Materials and Methods

2.1. Animals and Management

In this study, two experiments were performed: Experiment 1 and Experiment 2.

In Experiment 1, postpartum lactating Holstein dairy cows (n = 342) at two commercial dairy farms (A, B) in Hokkaido, northeast Japan, were used (parity: 2.3 ± 1.4, days in milk: 117.5 ± 55.2, previous AI number: 1.4 ± 1.6; mean ± SD). The cows selected for this study showed regular estrous cyclicity (estrous expression was observed 19–23 days after the previous estrous by farm staff) and were clinically healthy during the experimental period. The cows from farm A were kept in a free-stall barn, and those at farm B were kept in a tie-stall barn. The cows on both farms were fed a total mixed ration diet consisting of corn silage, grass silage, soybean meal, corn grain, and concentrate, with free access to water, and were milked twice daily. The mean annual milk production at farms A and B during the study period was 9300 kg and 12,500 kg, respectively. A voluntary waiting period was set at 40 days after parturition.

In Experiment 2, postpartum lactating Holstein dairy cows (n = 17) at the Fuji Animal Farm of Nippon Veterinary and Life Science University, located in Fujikawaguchiko-machi, Yamanashi, Japan, were used (parity: 4.8 ± 2.3, milk production: 41.3 ± 7.5 kg/d, days in milk on ovulation day: 94.0 ± 21.7 days; mean ± SD). All the cows calved spontaneously with no postpartum clinical diseases, such as retained placenta, metritis, ketosis, hypocalcemia, and displaced abomasum, from calving to the experiment. The cows were confirmed with regular estrus cyclicity and were clinically healthy during the experimental period; in addition, cows with AI numbers within two (first insemination: n = 12, second insemination: n = 5) were used in this experiment. The cows were housed in a tie-stall barn, milked twice daily, and had ad libitum access to water during the experiment. Lactating cows were fed 15 kg timothy hay, 3 kg alfalfa hay, 4 to 13 kg concentrate (16% DM CP), and 2.0 to 2.5 kg beet pulp. The concentrate was provided to the cows via an automatic concentrate feeder (Max Feeder HID, Orion, Nagano, Japan). The concentration and beet pulp were provided in accordance with each cow’s milk production.

2.2. Experimental Design

Experiment 1: This experiment was designed to evaluate the effect of each hormone treatment on reproductive performance. After the experiment had started, the first AI was conducted after spontaneous estrus. Farm staff detected spontaneous estrus by visual observation (standing heat, mounting to other cows, sniffing the vagina of other cows, swelling or hyperemia of the vagina, and mucous from the vagina), followed by rectal palpation of the genital tract performed by AI technicians or veterinarians to confirm the presence of a preovulatory follicle and a regressing CL (day of AI = Day 0). On Day 5, the cows were divided into the following three groups: I. a non-treatment group (n = 181), untreated; II. an IVPD group (n = 74), where a controlled internal drug release device (CIDR: CIDR 1900, Zoetis Japan, Tokyo, Japan) was inserted into the vagina from Day 5 to 19; and III. an hCG + IVPD group (n = 87), where 3000 IU hCG (Gestron 1500, Kyoritsu Seiyaku, Tokyo, Japan) was administered intramuscularly on Day 5 and an IVPD was inserted into the vagina from Day 5 to 19.

Re-estrus was detected by visual observation until the next estrus from 14 to 25 days after the first AI by farm staff. The estrus was confirmed by AI technicians or veterinarians by rectal palpation; then, the cows were artificially inseminated (second AI). Pregnancy diagnosis was determined by rectal palpation from 50 to 60 days after AI by veterinarians. If estrus was detected before the pregnancy diagnosis, the cows were determined to be non-pregnant.

The conception rate after the first AI was calculated by dividing the number of pregnant cows by the total number of cows enrolled in this study. The re-estrus detection rate was calculated by dividing the number of cows detected to be in re-estrus up to 25 days after the first AI by the number of non-pregnant cows. The conception rate after the second AI was calculated by dividing the number of pregnant cows by the number of cows detected to be in re-estrus up to 25 days after the first AI. Figure 1 shows a schematic diagram of the experimental model.

Experiment 2: This experiment was designed to evaluate the effects of no treatment, IVPD treatment, and hCG + IVPD treatment on plasma hormonal dynamics. Gonadotropin-releasing hormone (Fertirelin Inj Fujita, 100 μg, Fujita Pharmaceutical, Tokyo, Japan) was administered intramuscularly and an IVPD (CIDR, Zoetis Japan, Tokyo, Japan) was inserted into the vagina simultaneously on the first day. After 7 days, the IVPD was removed from the vagina, and PGF_2α (Pronargon F, 25 mg i.m., Zoetis Japan, Tokyo, Japan) was administered intramuscularly to induce luteolysis. Two days after PGF_2α treatment, GnRH was treated intramuscularly to induce ovulation. Thirty-six hours after the second round of GnRH administration, ovulation was confirmed using a transrectal ultrasonic device equipped with an 8.0-MHz linear transducer (EXAPADmini, IMV imaging, Angoulême, France) with the disappearance of the large follicles. We defined the day of ovulation as Day 1.

After confirming ovulation, the cows were assigned to the non-treatment (n = 6), IVPD (n = 6), or hCG + IVPD (n = 5) treatment group; each of the treatments was same as those in Experiment 1. Blood samples were collected into heparinized tubes (Venoject II, VP-H100K, Terumo, Tokyo, Japan) on Day 5, 8, 12, and 15 in all groups from the tail vein. Plasma was separated by centrifugation at 3000× g for 15 min at 4 °C and frozen at −30 °C until the determination of P₄ or E concentrations.

Plasma P₄ concentrations were determined in duplicate using a second-antibody enzyme immunoassay (EIA). All EIA procedures were as previously described [16]. Steroid hormone assays were performed after extraction with diethyl ether. The recovery rates of P₄ were 85%. The standard curve ranged from 0.05 to 50 ng/mL. The ED 50 of the assay was 2.4 ng/mL for P4. The intra- and interassay CVs were 4.2% and 6.6% at 2.0 ng/mL. Plasma E concentrations were determined in duplicate using a second-antibody enzyme immunoassay (EIA). All EIA procedures were as previously described [17]. Steroid hormone assays were performed after extraction with diethyl ether. All samples were concentrated 10 times for measuring. The recovery rate of E was 80%. The standard curve ranged from 2 to 2000 pg/mL. The ED50 of the assay was 58.0 pg/mL, and the intra- and interassay CV were 1.9% and 15.6% at 35 pg/mL.

2.3. Statistical Analysis

All statistical analyses were performed using EZR (version 1.50, Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a modified version of R commander designed to add statistical functions that are frequently used in biostatistics R (The R Foundation for Statistical Computing, Vienna, Austria) [18].

Multiple binary logistic regressions were conducted for analysis of the conception rate of the first AI, re-estrus detection rate, conception rate of the second AI, and cumulative pregnancy rate (proportion of pregnant cows from the first and second AIs combined). Pregnancy status or detected estrus was analyzed as a dependent variable (non-pregnant, 0; pregnant, 1; or non-detected, 0; detected, 1). The independent variables were treatment (non-treatment, IVPD, and hCG + IVPD), farm (A, B), AI number (≤3, >3), and interaction between treatment and AI number, and these were incorporated into the regression model. The cutoff between AI numbers ≤ 3 and >3 was selected based on findings from our previous study [9]. Parity and days in milk did not affect conception rate using IVPD treatment in the previous study [9]; thus, we did not include these factors as independent variables in this study. Our previous work has shown that the effect of IVPD treatment on conception rate was more pronounced in cows with >3 AIs. Therefore, we choose treatment, AI number, and interaction between treatment and AI number as independent variables.

Data on days to re-estrus from the first AI were confirmed to follow a normal distribution by the Shapiro–Wilk normality test and were analyzed by Bartlett’s test to verify the homogeneity of variances. The results of Bartlett’s test demonstrate that the variances in days to re-estrus from the first AI were not equal among the three treatment groups. Therefore, the Kruskal–Wallis test was used to determine the main effect of treatment (non-treatment vs. IVPD vs. hCG + IVPD).

We used a two-way ANOVA with repeated measures to determine the main effects of the group (non-treatment vs. IVPD vs. hCG + IVPD) and day (days from ovulation day) and their interaction to assess the sequential data of sex steroid hormones (plasma P₄ and E). When a group effect was detected with significance or a tendency, Holm’s test, as a multiple comparison test, was used to detect significant differences among the groups in terms of days.

A p-value of <0.05 indicated a significant difference, while a p-value <0.1 indicated a tendency.

3. Results

3.1. Effects of Treatment, Farm, AI Number, and Interaction of Treatment and AI Number on Conception Rate of First AI

Treatment (p < 0.01) and interaction of treatment and AI number (p < 0.001) were associated with the conception rate (

Table 1. Effects of intravaginal progesterone (P₄) device (IVPD) and hCG + IVPD treatment on the conception rate of the first AI.

Factor	Class	Conception Rate (%)	Odds Ratio	95% Confidence Interval	p-Value
Treatment	Non-treatment †	31.5 (57/181)	Reference
	IVPD ‡	48.6 (36/74)	1.33	0.68–2.61	0.411
	hCG + IVPD §	56.3 (49/87)	2.24	1.25–4.01	0.006
Farm	A	41.9 (103/246)	Reference
	B	40.6 (39/96)	0.77	0.47–1.29	0.322
AI number	≤3	40.3 (108/268)	Reference
	>3	45.9 (34/74)	0.43	0.17–1.13	0.086
Treatment × AI number	Non-treatment × ≤3	34.2 (51/149)	Reference
	Non-treatment × >3	18.8 (6/32)	0.44	0.17–1.15	0.093
	IVPD × ≤3	40.4 (19/47)	1.30	0.67–2.56	0.440
	IVPD × >3	63.0 (17/27)	3.27	1.39–7.65	0.006
	hCG + IVPD × ≤3	52.8 (38/72)	2.15	1.21–3.81	0.008
	hCG + IVPD × >3	73.3 (11/15)	5.28	1.60–17.40	0.006

^† Non-treatment: untreated after first AI. ^‡ IVPD: IVPD was inserted from 5 to 19 days after first AI. ^§ hCG + IVPD: 3000 IU hCG was administered intramuscularly at 5 days, and IVPD was inserted from 5 to 19 days after first AI.

). Conception rates in the hCG + IVPD group were significantly higher than that in the non-treatment group. Conception rates in the hCG + IVPD group, which underwent ≤3 AIs and >3 AIs, and the IVPD group, which underwent >3 AIs, were significantly higher than that in the non-treatment group, which underwent ≤3 AIs. Farm and AI number were not associated with the conception rate.

3.2. Effects of Treatment, Farm, AI Number, and Interaction of Treatment and AI Number on Re-Estrus Detection Rate up to 25 Days After First AI

Treatment (p < 0.01) was associated with re-estrus detection rate (

Table 2. Effects of intravaginal progesterone (P₄) device (IVPD) and hCG + IVPD treatment on re-estrus detection rate up to 25 days after first AI.

Factor	Class	Re-Estrus Detection Rate (%)	Odds Ratio	95% Confidence Interval	p-Value
Treatment	Non-treatment †	34.7 (43/124)	Reference
	IVPD ‡	78.9 (30/38)	5.06	2.00–12.80	0.0006
	hCG + IVPD §	60.5 (23/38)	2.69	1.19–6.08	0.017
Farm	A	43.4 (62/143)	Reference
	B	59.6 (34/57)	1.71	0.87–3.39	0.121
AI number	≤3	45.0 (72/160)	Reference
	>3	60.0 (24/40)	1.60	0.66–3.91	0.303

^† Non-treatment: untreated after first AI. ^‡ IVPD: IVPD was inserted from 5 to 19 days after first AI. ^§ hCG + IVPD: 3000 IU hCG was administered intramuscularly at 5 days, and IVPD was inserted from 5 to 19 days after first AI.

). Re-estrus detection rates in the IVPD and hCG + IVPD groups were significantly higher than that in the non-treatment group. Farm, AI number, and interaction of treatment and AI number were not associated with re-estrus detection rate.

3.3. Day to Re-Estrus from First AI in Non-Treatment, IVPD, and hCG + IVPD Groups in Non-Pregnant Cows

The mean, lower quartile, and upper quartile days to re-estrus were 22.0, 21.0, and 23.0 in the non-treatment group; 22.0, 21.0, and 21.5 in the IVPD group; and 22.0, 21.5, and 23.0 in the hCG + IVPD group, respectively. The treatment effect was not significant among the three groups.

3.4. Effects of Treatment, Farm, AI Number, and Interaction of Treatment and AI Number on Conception Rate of Second AI

Treatment was not associated with the conception rate of the second AI (

Table 3. Effects of intravaginal progesterone (P₄) device (IVPD) and hCG + IVPD treatment on conception rate of second AI at re-estrus up to Day 25 ^†.

Factor	Class	Conception Rate (%)	Odds Ratio	95% Confidence Interval	p-Value
Treatment	Non-treatment ‡	39.5 (17/43)	Reference
	IVPD §	33.3 (10/30)	0.35	0.04–3.02	0.339
	hCG + IVPD ¶	56.5 (13/23)	0.72	0.04–13.20	0.822
Farm	A	41.9 (26/62)	Reference
	B	41.2 (14/34)	0.97	0.40–2.35	0.950
AI number	≤3	40.3 (29/72)	Reference
	>3	45.8 (11/24)	2.28	0.56–9.25	0.248

^† Day 0 = day of first AI. ^‡ Non-treatment: untreated after first AI. ^§ IVPD: IVPD was inserted from 5 to 19 days after first AI. ^¶ hCG + IVPD: 3000 IU hCG was administered intramuscularly at 5 days, and IVPD was inserted from 5 to 19 days after first AI.

). No differences were observed among the IVPD or hCG + IVPD groups and the non-treatment groups. Farm, AI number, and interaction of treatment and AI number were not associated with the conception rate of the second AI.

3.5. Effects of Treatment, Farm, AI Number, and Interaction of Treatment and AI Number on Cumulative Pregnancy Rate

Treatment (p < 0.01) and interaction of treatment and AI number (p < 0.05) were associated with the cumulative pregnancy rate (

Table 4. Effects of intravaginal progesterone (P₄) device (IVPD) and hCG + IVPD treatment on cumulative pregnancy rate of first and second AI.

Factor	Class	Cumulative Pregnancy Rate (%)	Odds Ratio	95% Confidence Interval	p-Value
Treatment	Non-treatment †	40.9 (74/181)	Reference
	IVPD ‡	62.1 (46/74)	1.75	0.90–3.39	0.098
	hCG + IVPD §	71.3 (62/87)	3.63	1.67–5.53	<0.001
Farm	A	52.4 (129/246)	Reference
	B	55.2 (53/96)	0.91	0.55–1.51	0.711
AI number	≤3	51.1 (137/268)	Reference
	>3	60.8 (45/74)	0.84	0.38–1.84	0.656
Treatment × AI number	Non-treatment × ≤3	41.6 (62/149)	Reference
	Non-treatment × >3	37.5 (12/32)	0.84	0.38–1.85	0.668
	IVPD × ≤3	55.3 (26/47)	1.74	0.90–3.36	0.101
	IVPD × >3	74.0 (20/27)	4.01	1.60–10.10	0.003
	hCG + IVPD × ≤3	68.1 (49/72)	2.99	1.65–5.41	<0.001
	hCG + IVPD × >3	86.7 (13/15)	9.12	1.99–41.9	0.004

^† Non-treatment: untreated after first AI. ^‡ IVPD: IVPD was inserted from 5 to 19 days after first AI. ^§ hCG + IVPD: 3000 IU hCG was administered intramuscularly at 5 days, and IVPD was inserted from 5 to 19 days after first AI.

). The cumulative pregnancy rates in the IVPD and hCG + IVPD groups were significantly higher than that in the non-treatment group. The cumulative pregnancy rates in the hCG + IVPD group, which underwent ≤3 AIs and >3 AIs, and the IVPD group, which underwent >3 AIs, were significantly higher than that in non-treatment, which underwent ≤3 AIs.

Farm and AI number were not associated with the cumulative pregnancy rate.

3.6. Plasma P₄ and E Concentrations in Non-Treatment, IVPD, and hCG + IVPD Treatment Groups

The results are shown in Figure 2. For plasma P₄ concentration, the day effect was significant (p < 0.001) and the treatment effect showed a tendency (p = 0.051), and there were no differences in the treatment-by-day interaction. The plasma P₄ concentration in the hCG + IVPD group tended to be higher than that in the non-treatment group on Day 15. No differences were observed for day effect, treatment effect, and the treatment-by-day interaction in plasma E concentrations.

4. Discussion

The present study showed that hCG + IVPD treatment after AI significantly increased the conception rate of the first AI and cumulative pregnancy rate regardless of AI number. This treatment also increased re-estrus detection rates and did not affect the conception rate of the second AI in lactating dairy cows. In addition, the plasma P₄ concentration tended to be higher on Day 15 after hCG + IVPD treatment in lactating dairy cows with an early lactation period; this higher plasma P₄ concentration on Day 15 after AI may have resulted in higher conception rates in non-repeat breeder cows.

The results of our previous study showed that IVPD treatment 5 to 19 days after AI contributes to an increase in the conception rate only in repeat breeding lactating dairy cows (e.g., an AI number of more than three). IVPD treatment increases plasma P₄ concentration soon after the insertion of an IVPD from five to six days after AI [9]. An early increase in plasma P₄ concentration has been shown to improve embryonic development and hence the process of elongation, resulting in increased levels of interferon-tau to improve the maternal recognition of pregnancy mechanisms and reduce early embryonic death [19]. A previous report showed that the transfer of parthenogenetic embryos at Day 7 following AI to increase interferon-tau secretion increases the conception rate in repeat breeder lactating dairy cows [20]. This result shows that one of the reasons for infertility in repeat breeding cows may be insufficient levels of interferon-tau secretion from lower-quality embryos. On the other hand, hCG + IVPD treatment increased the conception rate of cows that underwent ≤3 AIs. One possible reason for this difference is that the hCG + IVPD treatment induced higher plasma P₄ concentrations in the later estrous cycle than the IVPD treatment, and this higher plasma P₄ concentration may have led to an increase in pregnancy maintenance due to the suppression of PGF_2α release during the maternal recognition period.

In a previous study, the plasma P₄ concentrations at seven days after IVPD treatment during the early luteal phase were not different from those under non-treatment conditions in lactating dairy cows [21]. In other words, IVPD treatment was unable to maintain high plasma P₄ levels until day seven post-insertion. Because of the formation of an accessory CL after hCG treatment, plasma P₄ concentrations are higher in the later luteal phase in heifers [14]. As in previous reports, hCG + IVPD treatment increased plasma P₄ concentration during the later estrous cycle in the present study. The time of pregnancy recognition, so called “the critical period”, may be defined to occur between Days 15 and 17 in cattle [22]. For maintaining pregnancy, an appropriate blockage of endometrial PGF_2α production to prevent luteolysis during the critical period is necessary to guarantee persistent CL graviditatis. Blocking of PGF_2α production was dependent on the ability of the production of interferon-tau from the embryo and appropriate condition of endometrium to respond to interferon-tau by the suppression of oxytocin receptors present on the endometrial cells [23]. Although sex steroid hormones affect the endometrium’s condition during the estrous cycle, factors that block the production of endometrial PGF_2α are decreased plasma estradiol-17β (E₂) [24] and increased P₄ concentration [25]. Although the plasma P₄ concentration was higher in the hCG + IVPD treatment, the plasma E concentration was not different in Experiment 2. Because the plasma P₄ concentration decreases with increasing milk production in lactating dairy cows [26], it was hypothesized that one of the causes of non-pregnancy in normal lactating dairy cows might the higher or ease of endometrial PGF_2α release caused by lower plasma P₄ concentrations during the critical period. Therefore, the level of plasma P₄ concentration could affect a more important role in maintaining pregnancy in this study. Taken together, hCG + IVPD treatment may enhance the plasma P₄ concentration from the early luteal phase to the critical period, and this leads to the higher conception rate at the first AI for lactating dairy cows that underwent ≤3 AIs in the present study. Further study will be needed to evaluate plasma P₄ concentrations during the IVPD and hCG + IVPD treatment periods in order to better explain the higher conception rate in the hCG + IVPD group, which underwent ≤3 AIs.

In the present study, re-estrus detection rates were significantly higher in the IVPD and hCG + IVPD treatment groups than in the non-treatment group. In our former study, IVPD treatment improved the re-estrus detection rate in lactating dairy cows [9]; this result is consistent with that of our present study. Estrus was highly expressed two to three days after IVPD removal in lactating dairy cows [9] and dairy heifers [26]; however, in the present study, the days to re-estrus from the first AI did not differ among treatments. Plasma P₄ concentrations prior to AI were associated with estrous intensity in both spontaneous estrus and synchronized AI programs in lactating dairy cows [27]; therefore, it has been speculated that P₄ supplementation by an IVPD may induce a higher intensity of re-estrus after the removal of the IVPD in non-pregnant cows. Further research is still needed to evaluate the effect of P₄ supplementation prior to the estrus.

Cumulative pregnancy rates were higher in the IVPD and hCG + IVPD groups than in the non-treatment group regardless of AI number. However, although IVPD treatment was not effective for increased cumulative pregnancy rate in cows that underwent less than four AIs, hCG + IVPD treatment increased the cumulative pregnancy rate regardless of AI number. This result showed that hCG + IVPD treatment 5 to 19 days after AI has a positive effect on fertility in cows that underwent less than four AIs. The cause of higher cumulative pregnancy rates from hCG + IVPD treatment in cows that underwent less than four AIs was the higher conception rate at the first AI.

5. Conclusions

The present study demonstrated that hCG administered five days after AI, combined with the insertion of an IVPD 5 to 19 days after AI, led to a greater conception rate, re-estrus detection rate, and cumulative pregnancy rate in lactating dairy cows. Therefore, hCG + IVPD treatment could be an effective treatment for improving the reproductive performance of normal lactating dairy cows.