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Subclinical endometritis is one of contributor factors of Repeat Breeder Syndrome. The aim of this study was to evaluate the effect of intrauterine cephapirin benzathine administration after timed artificial insemination (TAI) on the conception rate (CR) in repeat breeder dairy cows. All cows (n=335) had more than three services with no clinically abnormalities of the reproductive tract were received the combined protocol; An ear implant containing progestagen added the Ovsynch protocol plus third GnRH administration 7 d after TAI. Cows in the treatment group (TRT; n=160) were administered intrauterine cephapirin 12 h after TAI and untreated cows served as a control group (CON; n=175). Synchronization rate (98.8 % in TRT, 97.1 % in CON) and response to third GnRH (88.1% in TRT, 83.9% in CON) were similar between groups. There was no effect of cephapirin on 31 and 62 days CR in repeat breeder cows (43.8% in TRT and 44.0% in CON; 39.4% in TRT and 40.6% in CON, respectively). Thus, the results showed that there was no effect of post AI cephapirin treatment on improving CR, however its thought that the combined protocol was used in this study may be useful for repeat breeder cows.
Keywords: Repeat breeder, conception rate, endometritis, cephapirin
Repeat breeder syndrome is a major economic loss in dairy industry due to more insemination cost, increased calving interval and increased culling rates (Bartlett et al. 1998). Cows that fail to conceive after more than three inseminations with fertile semen in the absence of detectable abnormalities are classified as repeat breeders (Zemjanis 1980). Besides the causes of the repeat breeding are multifactorial, it is likely that subclinical endometritis is an important contributor to the repeat breeder syndrome of bovine subfertility (Noakes et al. 2001). Endometritis is one of the most important causes of abnormal uterine environment and especially subclinic endometritis results with repeat breeding. The presence of pathogenic bacteria in the uterus causes inflammation, histological lesions of the endometrium, distrupted embryo survival (Sheldon et al. 2006). Therefore improvement of the intrauterine environment for embryo survival is the one of different therapeutic methods for repeat breeding.
Treatment of bacterial endometritis, with intrauterine infusion of antibacterial agents and antibiotics, before or after insemination, resulted in varying degrees of success (Veselinovic et al. 1996; Shams-Esfandabadi et al. 2004; Kasimanickam et al. 2005; Ahmadi and Dehghan 2007). Cephapirin, a first-generation cephalosporin antibiotic, is active against Gram-positive organisms and anaerobic bacteria, and less active against Gram-negative organisms. For this reason Cephapirin appeared to be rational antibiotic choice for intrauterine infusion (Mc Dougall 2001; LeBlanc et al. 2002). Kasimanickam et al. (2005) reported that a single treatment with cephapirin significantly improved the reproductive performance of cows with subclinical endometritis. Veselinovic et al. (1996), reported that cephapirin is sufficient when applied once in case of clinical endometritis with low degree. Thus, the aim of this study was to evaluate the effect of post-AI Cephapirin benzathine (cephapirin) administration on conception rate in repeat breeder dairy cows.
MATERIAL AND METHODS
Cows, housing and management
This study was conducted on a commercial dairy herd (approximately 800 lactating dairy cows) in South Marmara region, Bursa, Turkey. Herd composition was 2/3 Holstein-Friesian and 1/3 Swedish-Red cows. The lactating cows were housed in free stall facilities and grouped according to their milk production. All cows were milked three times a day and fed complete mixed rations based on NRC recommendations (NRC 2001). Mean milk production of the herd was 9.880 kg (305 d) per cow. Daily milk yield was collected by the ALPRO™ system (DeLaval, Sweden). Average milk production for each cow was recorded for 7 d before and after AI. All cows had their body condition scored using a 5-point (1=thin to 5=fat) scoring system (Ferguson et al. 1994). All animal handling and procedures were approved by the Lalahan Livestock Central Research Institute Animal Care Committee.
Synchronization protocol and treatment
A total of 335 cows (283 Holstein-Frisian and 52 Swedish-Red cows) were used in this study. Cows had more than three inseminations with no clinically abnormalities of the reproductive tract selected as repeat breeder in this study. All cows were received the same timed artificial insemination (TAI) protocol -the Ovsynch- added progestagen and administered GnRH seven days after TAI. Cows, at random stages of the estrous cycle, received an ear implant containing progestagen (3 mg Norgestomet, Crestar, Intervet, Turkey) and GnRH administration (10 µg Busereline acetate, Receptal, Intervet, Turkey), on day 0 and PGF2α administration (500 µg Cloprostenol, Estrumate, CEVA-DIF, Turkey) at the time of implant removal, on day 7 and second GnRH injection, on day 9. Then the cows artificially inseminated, at fixed time, 16-18 h after the second GnRH treatment, using frozen-thawed semen from bulls of proven fertility by farm veterinarians. All inseminated cows received third GnRH administration (10 µg Busereline acetate, Receptal) seven days after AI, on day 17.
All inseminated cows were randomly divided into two groups. The cows in the treatment Group (TRT) were recevied intrauterine 500 mg of cephapirin benzathine (Metricure, Intervet, Turkey) 12 h after TAI; the cows in the Control Group (CON) were not received any treatment after TAI. The experimental protocol is summarized in Figure 1.
Cows' ovaries were examined on the day of first GnRH and ear implant administrations and 7 days later to determine ovulation response to first GnRH treatment by ultrasound machine (Honda HS 2000 equipped with a 7.5 MHz transducer, Honda, Japan). Response to first GnRH was characterized by appearance of a new corpus luteum (CL) on ovaries. Maximum follicular size was measured at the time of AI. Cows were examined seven days after TAI to determine ovulation by disappearing of dominant follicle at time of AI and appearence of new CL and also examined to measure follicle size before third GnRH treatment. Response to the third GnRH administration was detected 7 days later third GnRH application (Figure 1).
Pregnancy diagnosis was performed 31 days post-insemination (Visualization of a fluid-filled uterine horn with embryonic vesicles) by ultrasonography. Pregnancy check was done post insemination 62 days (Presence of a fetus). Pregnancy loss was recorded when the second check founded negative. Conception rate was calculated as the number of cows diagnosed pregnant, divided by the number of cows receiving AI.
All statistical procedures were performed using the computational software of SAS (Release 9.2, SAS Institute, Cary, NC). For statistical analyses, Breed of lactating cows was coded as 1 (Holstein-Friesian) or 2 (Swedish-Red). The ovulatory response to first, second and third GnRH administration was coded as 0 (No) or 1 (Yes). Presence of conception on d 31 and 62 after insemination was coded as 0 (Not pregnant) or 1 (Pregnant). Primiparous and multiparous cows were coded as 1 or 2, respectively.
GLM procedure was performed in order to analyze the following; to compare milk production, days in milk (DIM), body condition score (BCS), parity, number of services and follicle size at the time of AI and at the time of third GnRH between groups; to determine the effect of synchronization protocol, parity and response to first and third GnRH on follicle size at the time of AI. Chi-square analysis using the PROC FREQ procedure was used to analyze the following: to compare of ovulatory response to first, second and third GnRH and conception rate (CR) at 31. and 62. days. Logistic procedure was used to analyze the following: the effect of treatment, milk production, DIM, BCS, follicle size at the time of AI, response to first and third GnRH and breed on conception rate.
Mean lactation number of cows was similar between groups (2.1±0.07 in TRT and 2.2±0.06 in CON, respectively). The mean DIM did not differ between TRT and CON groups, 260.3±6.6 and 269.5±6.4, respectively. BCS was found to be similar between TRT and CON groups (2.92±0.01 and 3.01±0.03, respectively). But average milk production was different (P=0.03) between groups (31.4±0.6 kg/d in TRT and 29.5±0.6 kg/d in CON). Number of services was not different between groups (3.9±0.1 in TRT and 4.1±0.09 in CON).
Percentage of cows that ovulated in response to the first GnRH treatment did not differ in TRT (67.5%, 108/160) and CON (70.9%, 124/175) groups (Table 1). Synchronization rate (response to second GnRH) was not different between groups (98.8%, 158/160 in TRT and 97.1%, 170/175 in CON) (Table 1). Response to the third GnRH was similar between groups (89.2%, 141/158 in TRT and 82.9%, 141/170 in CON) (Table 1).
Maximum follicular size at the time of AI did not differ between TRT (16.07±0.18 mm) and CON (16.15±0.18 mm) groups (Table 2). However, cows that ovulated in response to first GnRH of Ovsynch had produce smaller (P=0.01) follicle size at the time of AI (15.8±0.15 mm) than those of non-responsive (16.5±0.23 mm). Presence of accesory corpus CL at the 31 and 62 days pregnancy examinations was similar in groups (Table 1).
Effect of cephapirin on CR; at 31 d was similar between treatment groups with 43.8% (70/160) in TRT and 44.0% (77/175) in CON. When 62 d CR were evaluated in groups, there was no differences between TRT (39.4%; 63/160) and CON (40.6%; 71/175) groups (Table 3). Milk production, DIM, BCS, parity, breed, response to first, second and third GnRH administrations, follicle size at the time of AI and post AI cephapirin treatment did not effect on CR.
Repeat breeder syndrome is a multifactorial condition and there is no accurate method for diagnosing the cause of most individual cases. Eventhough there are many causative factors; hormonal imbalance and uterine infection are considered as important etiological factors for repeat breeder cows (Zemjanis 1980). In this study, the combined protocol (the Ovsynch added progestagen plus GnRH administration 7 d after AI) was used for eliminating etiologic hormonal and management factors as much as possible in repeat breeder cows; like anovulation, delayed ovulation, progesterone deficiency, estrus detection errors etc.
Recent studies results showed that when only Ovsynch protocol was applied to normal lactating dairy cows, ovulatory response to first GnRH of Ovsynch was detected between 45 and 95% and synchronization rate was determined between 75 and 90% (Pursley et al. 1995; Galvão and Santos 2008). Stevenson et al. (2006) reported that synchronization rate after Ovsynch+CIDR as 85-95%. In this study, first ovulatory response and synchronization rate after combined protocol with Ovsynch+progestagen in repeat breeder cows produced similar results with previous studies (Gumen et al. 2003; Bello et al. 2006). In addition, earlier studies indicated that conception rate was 30 to 45% after Ovsynch in normal dairy cows (Pursley et al. 1995; Bello et al. 2006). In this study, conception rate was found 44.0% in CON and 43.8% in TRT groups. This results indicate that combined protocol used in this study improved fertility in repeat breeder dairy cows.
When the responses of the groups to this combined protocol evaluated, response to first GnRH, synchronization rate, response to third GnRH and the follicle size at the time of AI were similar between the treatment and the control groups (Table 1 and 2). Also, there were any differences between groups related with covariant factors as DIM, BCS, number of services and parity. Similarity of the groups at responses to the combined protocol and covariant factors, make the evaluation of the effect of post-AI cephapirin administration in repeat breeder cows more clear.
One of the leading factors to repeat breeding with a high incidence is metritis and different degrees of endometritis due to invading of specific and non-specific infectious agents to uterus (Hartîgan et al. 1972). Its known that subclinical endometritis especially at artificial insemination might impair the uterine environment and disrupt the implantation and development of the embryo (Kaufmann et al. 2009). Therefore, evaluation the attempts of the elimination of endometritis with several intruterine antibiotic infusions at different periods in repeat breeder or normal cows has been the objective of several studies (Veselinovic et al. 1996; Kasimanickam et al. 2005).
Besides post-AI intrauterin antibiotic administration is well known in the field, we could achieve only a few scientific paper evaluated the effect of post-insemination intrauterin antibiotic administration on endometritis (Gupta et al. 1983; Ozturkler et al. 2001; Shams-Esfandabadi et al. 2004). In these limited studies, similiar to our results, some authors did not found any beneficial effect of intrauterine antibiotic treatment like our study (Gupta et al. 1983; Shams-Esfandabadi et al. 2004). Although Ozturkler et al. (2001) did not mentioned the effect of intrauterin antibiotic treatment using alone, they found that hCG plus intrauterine antibiotic administrations after insemination beneficial to improve fertility in repeat breeder cows.
Cephapirin, appeared to be rational antibiotic choice for intrauterine infusion with activity against Gram-positive organisms and anaerobic bacteria, with no witholding period for milk. In this study, we thought that a single dose of post-AI intrauterine cephapirin administration can improve the CR in repeat breeder lactating cows due to eliminating subclinic endometritis without any milk waste. Kasimanickam et al. (2005), reported that a single treatment with cephapirin significantly improved the reproductive performance of cows with subclinical endometritis. Nevetheless, in the current study, Cephapirin administration did not enhanced CR in repeat breeder cows. Similarly, in the study of Ahmadi and Dehghan (2007) resulted with uneffectiveness of cephapirin administration, they purposed that uterine lavage plus PGF2α administration, without cephapirin, may be preferable in the treatment of repeat breeder cows. Also there are studies suggested that PGF2α administration is more (Steffan et al. 1984) or equally (Sheldon and Noakes 1998; Kasimanickam et al. 2005). effective than intrauterine antibiotics for the improvement of reproductive performance in cows with endometritis.
Our results showed that post-AI intrauterine cephapirin administration did not enhance conception rate in repeat breeder dairy cows. Additionally, combined synchronization protocol had been used in this study; the progestagen added Ovsynch protocol plus GnRH 7 days after AI, improved CR spectacularly in repeat breeder dairy cows.
Table 1. Responses to the combined synchronization protocol at repeat breeder dairy cows.
The Treatment Group
The Control Group
Response to first GnRH of Ovsynch
Response to second GnRH of Ovsynch
Response to third GnRH (7 d after AI)
Presence of accessory CL
at 31 days pregnancy
Presence of accessory CL
at 62 days pregnancy
Table 2 Maximum follicle size at the time of AI and third GnRH after TAI and difference of follicle size in cows that respond to first GnRH or not in repeat breeder dairy cows.
The Treatment Group
The Control Group
Follicle size at the time of AI
Follicle size at time of
Ovulatory Response (+)
Ovulatory Response (-)
Maximum follicle size
15.8±0.1 a mm
16.5±0.23 b mm
Table 3 Conception rate after combined protocol at 31 and 62 d post-insemination in repeat breeder dairy cows.
The Treatment Group
The Control Group
Pregnancy rate at 31 d
Pregnancy rate at 62 d
Figure 1. Summary of experimental design showing combined protocol and cephapirin benzathine (cephapirin) treatment in repeat breeder cows. Combined protocol used to eliminate any possible hormonal problems in repeat breeder cows. The protocol consist of Ovsynch plus an ear implant (3 mg Norgestomet) for 7 d between GnRH-PGF2α and GnRH treatment 7 d after insemination to enhance serum progesterone level by causing accessory CL. Intrauterine cephapirin were used 12 h after insemination in the treatment group (TRT) and untreated cows served as an control (CON).