The Association of Some Variables with First Service Conception Rate Using Modified-Cosynch and G6G Protocols in Cows

Different hormonal programs have been developed to implement estrous induction and a Timed Artificial Insemination (TAI) in lactating dairy cows. Estrous induction and synchronization were done using the analogous of reproductive hormones found within the hypothalamus, ovary and uterus [1]. These strategies optimized reproductive efficiency by regulation of ovarian function [2]. Synchronized estrus and/or ovulation at the end of all reproductive hormonal programs were needed to increase efficiency of protocols. Programs that let us know when ovulation happens were developed in the 1990s [1]. They have been necessary for taking optimum conception rate followed by estrous induction and especially for postpartum first timed artificial insemination. The time of ovulation was controlled to increase conception rate followed by postpartum first TAI. Timed artificial insemination protocols were used to induce fertile ovulation without estrous expression [2,3]. So, the use of TAI has become an integral part of reproductive management in dairy herds with poor and inadequate estrous detection [4]. Protocols were used to synchronize corpus luteum regression, follicle ovulation and they brought about TAI resulting in improved reproductive performance [4,5]. In this regard, many protocols such as G6G and Cosynch have been adopted in many dairy herds [6,7]. The above protocols are adopted in some dairy herds that enrolled cows in a controlled breeding program when they become eligible for insemination [1].

Others which use high hormonal intervention start them at clean test. The Ovsynch protocol for Timed Artificial Insemination (TAI) is widely used, although, some limitations of the ovsynch program were reported [7]. Limitations include acceptable level of progesterone at the time of TAI [8] and inability of single dose of PGF2α to induce complete luteolysis [8]. G6G protocol reduces these limitations and allows maximum ovulatory response to first GnRH injection of Ovsynch and greater ovulatory response to 2nd GnRH injection [9]. The logic behind Cosynch protocol was to reduce the number of handlings, so it is often preferred, as TAI and the second GnRH injection may be conducted simultaneously. In recent studies, practical short-term synchronization was reported [10,11]. Therefore, the present study was designed to evaluate the first service conception rate of cows after using two protocols include short-term Modified-Cosynch and G6G protocols regarding various factors including calving to first AI interval, different seasons of AI and insemination based on the estrous detection or timed artificial insemination.

Animals, housing and management

The study was carried out in a large commercial dairy farm near Shiraz, Fars province, in central Iran (29°58′34″N, 52°40′45″E). This study was approved by the Ethics Committee of Shiraz University. We used the two hormonal protocols in cows with corpus luteum (n=503) for postpartum first service from 47 to 80 days in milk after calving. The mean (±SD) of days in milk of studied cows was 61.9±8.2. The farm milked 1900 Holstein cows three times daily. The cows were housed in free stall barns with sand bedding. Cows calved throughout the year, and the herd had annual average milk yields of above 9000 liters per cow. Cows received corn silage, alfalfa hay and concentrates (containing corn meal, soybean meal, vitamins and minerals). They were maintained in a close-up dry group for 3 weeks before calving. The cows calved in an open-shed barn. Fresh cows were kept in a transition group for 20 days.

Study design

The cows were examined by transrectal ultrasonography and ovarian structures determined by presence or absence of Corpus Luteum (CL) and only cows were used with corpus luteum. The following protocols were used for different groups and are shown in Figure 1. Group G6G (n=217): administration of 500μg of a synthetic analogue of prostaglandin F2α (Estron, Cloprostenol 0.25mg/ml; Bioveta, Czech Republic) on day 0; administration of 100μg of a synthetic analogue of gonadotropin-releasing hormone (Gona breed, Gonadorelin (as acetate) 100μg/ml, Parnell lab, New Zealand) on day 2 and repeat that on day 8. Day 15: administration of 500μg of a synthetic analogue of prostaglandin F2α, 56h after PG injection: administration of 100μg of a synthetic analogue of gonadotropin-releasing hormone. Cows were inseminated at any time after PGF2α upon detection of estrus, and others that didn’t show estrous sings received Timed Artificial Insemination (TAI) 16h after gonadotropin-releasing hormone (GnRH).

Fgure 1:Study design of the treatment protocols. Cows received Timed Artificial Insemination (TAI) 16h after Gonadotropin-Releasing Hormone (GnRH) or 32h after Estradiol Benzoate (EB) treatment. Some cows were inseminated at any time after PGF2α or EB upon heat detection.

Group Modified-Cosynch or short-term Modified-Cosynch (n=286): administration of 500μg of a synthetic analogue of prostaglandin F2α on day 0, 48h after PG injection: administration of 1mg of estradiol benzoate (Vetastrol, Aburaihan Pharmaceutical Co; Iran) and the cows were inseminated with estrous signs or timed artificial insemination was done on 32 hours after receive Estradiol Benzoate (EB). At the time of AI, administration of 100μg of a synthetic analogue of gonadotropin-releasing hormone was done. In this study, oestrous detection was performed 3 times a day for a period of 20min each at about 4-6am; 4-6pm and 8-12pm. Cows that showed standing heat were artificially inseminated using frozen semen of proved fertility 12h later. All cows were not considered to be in heat underwent the Timed Artificial Insemination (TAI) 16h after the second GnRH dose or 32h after EB injection. Pregnancy was diagnosed by transrectal ultrasonography (5MHz rectal linear probe, BCF Company, UK) from 31 to 35 days after insemination.

Statistical analysis

All analyses were performed using the SPSS statistical software (version 23.0, SPSS, Inc; Chicago, IL, USA). Chi-square test was used to compare the numbers and the proportions of pregnant cows regarding various factors including calving to first AI interval (≤60 and >60DIM), two hormonal protocols, different seasons of AI (winter vs other seasons) and insemination based on the estrous detection (HAI) or Timed Artificial Insemination (TAI). Possible effects of insemination time after calving (days in milk, DIM), hormonal protocols, insemination season and type (TAI or HAI) on the conception rate were explored using logistic regression analysis. The final models were assessed using the Hosmer-Leme show goodness of fit test that reported in the results. The data from each studied group were compared by means of logistic regression analysis using pregnancy as the dependent variable (0 denotes not pregnant and 1 denotes pregnant) and season, insemination type, used hormonal protocols and calving to insemination interval or DIM as independent factors. We used the data of inseminated cows with G6G protocol, on day ≤60 after calving, in winter, and based on the timed AI (TAI) was used as reference and the model fits. Days in milk and hormonal protocols that had been selected in those stepping approaches entered the final Likelihood Ratio (LR), in which the final odds ratio estimates with 95% confidence intervals were derived. The results are expressed as percentages, and the probability values of P≤0.05 are considered significant.

The number (percentage) of cows showed standing heat (estrus) was 238 (47.3%) and the rest consisting of 265 cows (52.7%) didn’t show heat and underwent Timed Artificial Insemination (TAI). Conception rates were 32.8% (78/238) and 28.3% (75/265) for inseminated cows based on the heat detection and TAI, respectively (P=0.2). Conception rates of studied cows based on the type of artificial insemination and postpartum days in milk, season and hormonal protocols were shown in Tables 1-3. The numbers and the proportions of conceived cows regarding hormonal protocols, days in milk and season AI were shown in Table 1. The conception rate was greater (P=0.02) for short-term Modified-Cosynch than that for G6G group (34.6 vs 24.9%). Higher conception rates (35.6%; P=0.004) were found for inseminated cows during >60 DIM (Table 1). Inseminated cows after Modified- Cosynch had higher conception rate than those cows after G6G protocol in winter (40 vs 24.7%; P=0.02; Table 2). The conception rate of cows studied after TAI or HAI was higher in inseminated cows >60 days in milk that cows were inseminated ≤60 DIM (33.1 vs 22.5% and 38.1 vs 25.3%, respectively; Table 3). Conception rate of cows was inseminated underwent TAI was higher in winter compared with other seasons (39.3 vs 23.2%; P=0.007; Table 3). Possible effects of risk factors on the conception rate of studied cows were explored using logistic regression analysis and shown in Tables 4. Inseminated cows at >60 DIM had significantly higher odds of conceiving (OR=1.8) than the ≤60 DIM (P=0.004, Table 4). Higher odds of conceiving (OR=1.6) were found for inseminated cows after short-term Modified-Cosynch compared with the cows after G6G protocol (P=0.02).

Table 1:First service conception rate of studied cows after using protocols during early lactation.

Table 2:First service conception rate (%) of studied cows after using hormonal protocols in different days in milk and seasons.

a, b Different superscripts indicate significant differences within columns (P<0.05).

Table 3:First service conception rate (%) of studied cows after Timed Artificial Insemination (TAI) or insemination after heat detection (HAI) in different days in milk and seasons.

a, b Different superscripts indicate significant differences within columns (P<0.05).

Table 4:Odds ratios of the variables included in the final logistic regression model for conception rate.

Likelihood ratio test=8.9, 2df, P=0.01; Hosmer and Lem show goodness-of-fit test=0.25, 2df, P=0.8; The model fits.

The results of this study indicate that the first service conception rate was higher in cows with CL that were subjected to short-term modified-Cosynch. There was a higher conception rate for inseminated cows at >60 postpartum days compared with those inseminated earlier. A higher conception rate of cows was found in winter compared with other seasons. Cosynch is a timed-insemination program the same as Ovsynch protocol, with one exception, AI was done at the time of the final GnRH injection [12,13]. In the present study, we used short-term Modified-Cosynch protocol; the cows were inseminated with estrous signs or timed artificial insemination was done on 32 hours after receiving EB. We administered gonadotropin-releasing hormone at the time of AI. In this study, we used the short-term modified-Cosynch protocol for reducing the number of handlings, so it was preferred. In addition, while cows were subjected to short-term modified-Cosynch, the acceptable conception rate was found. Another protocol (G6G) needs to give injections on too many different days and requires consistency in assuring in injections are given on time and at the right time. Short-term modified-Cosynch protocol increases conception rate of inseminated cows compared with G6G protocols. Similar pregnancy outcomes after TAI were reported for the G6G program in Holstein cows by Khalil AAY [14].

The contraception rate of inseminated cows after G6G protocol was lower than that recorded (32.9%) by Heidari F et al. [15]. The day of the estrous cycle and postpartum days in milk when the treatment initiated could be the cause of these conflict results in different studies. Although, in this study, similar conception rates were obtained after insemination of cows based on the estrus in different seasons, they were higher in winter for inseminated cows based on the TAI regardless of the hormonal protocols. Khalil AAY [14] found that the cows bred during the cold season had higher pregnancy rate compared with those bred during the hot season after TAI. Pregnancy rate of treated cows with PGF and EB 24h or GnRH 56h apart was greater after TAI in winter [10]. Stevenson and Pulley [16] reported that the pregnancy rate of cows treated with PG-3-G before ovsynch-56 TAI protocol was higher than inseminating cows at estrus during cooler weather. Santos VG et al. [17] reported the higher first postpartum submission rates for cows received TAI compared with cows inseminated after a detected estrus during 70-84 DIM. They also found more pregnancy per AI in the former group. It is possible that the increase in embryo quality and timing of insemination relative to ovulation result in more pregnancy per AI [18-20].

The cows inserted in cold (42.2%) season had greater pregnancy rate than those inseminated during hot season (29.8%) after G6G and Presynch-10-ovsynch protocols [16]. Yamada K [21] reported that the conception rate of cows after ovsynch/ TAI increased in winter in Japan (52.3-55.6%). In this study, we found that these protocols could be used in winter with 34.2% conception rate. In the present study, significant differences were detected in conception rate between the inseminated cows after TAI or HAI in different days in milk. Stevenson and Pulley [16] reported that the pregnancy rate of cows identified in estrus on the day of insemination was higher compared with those not detected in estrus on the day of TAI after G6G and Presynch-10-ovsynch protocols (39.3% vs 32.4%). The first service conception rate for observed heat artificial insemination cows was higher compared with TAI cows (35.3% versus 21.0%) in ovsynch synchronization protocol [22]. A variety of strategies using an activity-monitoring system and TAI for postpartum first AI was recommended by Fricke et al. [23]. A meta-Analysis by Borchardt et al. [24] underlined the importance of ovarian physiology to maximize fertility in TAI protocols. The conception rate of timed AI cows was the lowest from 40 to 60 (47.8%) and the highest from 101 to 120 (54.7%) days postpartum after ovsynch/TAI protocol [21]. It is assumed that first postpartum delayed ovulation might have been one of the causes of the low conception rate for inseminated cows during 40 to 60 DIM. The high conception rate of cows had corpus luteum and normal ovarian activity was reported [21].

In the current study, cows inseminated later in DIM had higher odds of conceiving than the reference category (early insemination). Higher odds of conceiving were also found in cows that were subjected to modified-Cosynch compared with the reference category (G6G). It has been reported that the treated cows with PGF and EB 24h apart had greater pregnancy rates after TAI compared with those received PGF and GnRH 56h, when a functional CL was accurately detected [10]. The foundation hormone of any protocol is PGF2α and it is very effective management tool if most cows are cycling. Development of practical protocols using palpation per rectum may be necessary where the cost of the hormone is high. It seems clear that a high conception rate can be achieved if proper synchronization protocols are used [25]. Simplification of the protocols is necessary to improve reproductive management in dairy farms. However, if the G6G protocol has been selected for routine use, the number of injections has also been considered.

This study may make use of suitable protocols for first postpartum inseminations about the days in milk of cows, breeding season and type of insemination in modern dairy farming. The days after calving, season and type of insemination should be considered when selecting the appropriate program for postpartum inseminated cows.

The authors thank the owners and staff of the Pegah Fars milk company for their cooperation (Mehdi Alipour and Samira Iranpour). The authors intend to thank Mohammad Reza Mirzaei for editing the final draft of the manuscript. The authors also thank Shiraz University for financial support (grant no. 97GCU2M1251).

1. Lucy MC, McDougall S, Nation DP (2004) The use of hormonal treatments to improve the reproductive performance of lactating dairy cows in feedlot or pasture-based management systems. Anim Reprod Sci 82-83: 495-512.
2. Thatcher WW, Moreira F, Pancarci SM, Bartolome JA, Santos JE (2002) Strategies to optimize reproductive efficiency by regulation of ovarian function. Domest Anim Endocrinol 23(1-2): 243-254.
3. Diskin MG, Austin EJ, Roche JF (2002) Exogenous hormonal manipulation of ovarian activity in cattle. Domest Anim Endocrinol 23(1-2): 211-228.
4. Colazo MG, Mapletoft RJ (2014) A review of current timed-AI (TAI) programs for beef and dairy cattle. Can Vet J 55(8): 772-780.
5. Selvaraju M, Velladurai C, Alagar S (2020) Fixed time breeding programs in cows, buffaloes, goats and sheep: A review. Pharm Innov J 9(9): 77-83.
6. Colazo MG, Ambrose DJ (2013) New research in controlled breeding programs for dairy cattle. WCDS Adv Dairy Technol 25: 75-95.
7. Nowicki A, Barański W, Baryczka A, Janowski T (2017) OvSynch protocol and its modifications in the reproduction management of dairy cattle herds-an update. J Vet Res 61(3): 329-336.
8. Santos JE, Narciso CD, Rivera F, Thatcher WW, Chebel RC (2010) Effect of reducing the period of follicle dominance in a timed artificial insemination protocol on reproduction of dairy cows. J Dairy Sci 93(7): 2976-2988.
9. Yousuf MR, Martins JPN, Ahmad N, Nobis K, Pursley JR (2016) Pre synchronization of lactating dairy cows with PGF2α and GnRH simultaneously, 7 days before Ovsynch have similar outcomes compared to G6G. Theriogenology 86(6): 1607-1614.
10. Bandai K, Kusaka H, Miura H, Kikuchi M, Sakaguchi M (2020) A simple and practical short-term timed artificial insemination protocol using estradiol benzoate with prostaglandin F2α in lactating dairy cows. Theriogenology 141: 197-201.
11. Sauls-Hiesterman JA, Voelz BE, Stevenson JS (2020) A shortened resynchronization treatment for dairy cows after a nonpregnancy diagnosis. Theriogenology 141: 105-112.
12. Caraba IV, Velicevici S (2013) Using ovsynch protocol versus cosynch protocol in dairy cows. Sci P Anim Sci Biotechnol 46(2): 63-65.
13. Meglić P, Špoljarić B, Štibrić G, Samardžija M, Lojkić M, et al. (2023) Ovsynch based protocols in reproductive management and infertility treatment in dairy cows-when and why? Vet Stanica 54(2): 213-22.
14. Khalil AAY (2019) Fertility response of lactating dairy cows subjected to three different breeding programs under subtropical conditions. Beni-Suef Univ J Basic Appl Sci 8(6): 1-10.
15. Heidari F, Dirandeh E, Pirsaraei ZA, Colazo MG (2017) Modifications of the G6G timed-AI protocol improved pregnancy per AI and reduced pregnancy loss in lactating dairy cows. Animal 11(11): 2002-2009.
16. Stevenson JS, Pulley SL (2012) Pregnancy per artificial insemination after presynchronizing estrous cycles with the Presynch-10 protocol or prostaglandin F2α injection followed by gonadotropin-releasing hormone before ovsynch-56 in 4 dairy herds of lactating dairy cows. J Dairy Sci 95(11): 6513-6522.
17. Santos VG, Carvalho PD, Maia C, Carneiro B, Valenza A, et al. (2017) Fertility of lactating Holstein cows submitted to a double-ovsynch protocol and timed artificial insemination versus artificial insemination after synchronization of estrus at a similar day in milk range. J Dairy Sci 100(10): 8507-8517.
18. Wiltbank MC, Sartori R, Herlihy MM, Vasconcelos JLM, Nascimento AB, et al. (2011) Managing the dominant follicle in lactating dairy cows. Theriogenology 76(9): 1568-1582.
19. Wiltbank MC, Souza AH, Carvalho PD, Bender RW, Nascimento AB (2011) Improving fertility to timed artificial insemination by manipulation of circulating progesterone concentrations in lactating dairy cattle. Reprod Fertil Dev 24(1): 238-243.
20. Valenza A, Giordano JO, Lopes G, Vincenti L, Amundson MC, et al. (2012) Assessment of an accelerometer system for detection of estrus and treatment with gonadotropin-releasing hormone at the time of insemination in lactating dairy cows. J Dairy Sci 95(12): 7115-7127.
21. Yamada K (2005) Development of ovulation synchronization and fixed time artificial insemination in dairy cows. J Reprod Dev 51(2): 177-186.
22. Kasimanickam R, Cornwell JM, Nebel RL (2005) Fertility following fixed-time AI or insemination at observed estrus in Ovsynch and Heat synch programs in lactating dairy cows. Theriogenology 63(9): 2550-2559.
23. Fricke PM, Giordano JO, Valenza A, Lopes JrG, Amundson MC, et al. (2014) Reproductive performance of lactating dairy cows managed for first service using timed artificial insemination with or without detection of estrus using an activity-monitoring system. J Dairy Sci 97(5): 2771-2781.
24. Borchardt S, Pohl A, Heuwieser W (2020) Luteal presence and ovarian response at the beginning of a timed artificial insemination protocol for lactating dairy cows affect fertility: A meta-analysis. Animals 10(9): 1551.
25. Giordano JO, Wiltbank MC, Fricke PM, Bas S, Pawlisch R, et al. (2013) Effect of increasing GnRH and PGF2α dose during double-ovsynch on ovulatory response, luteal regression and fertility of lactating dairy cows. Theriogenology 80(7): 773-783.