Risk Factors Influencing Conception Rate in Holstein Heifers Before Artificial Insemination or Embryo Transfer

The objective of this study was to show the risk factors affecting the conception rate in Holstein heifers after synchronization of estrus. A total of 275 Holstein heifers housed in a free barn were used for the experiment. The herd was visited regularly at four week intervals for synchronization of estrus using Heatsynch and CIDR-Heatsynch protocols. A group of four to 14 animals, depending on the availability, were referred to the experiment at each visit. Estrus induction rates in the two protocols were 93.9% and 94.9%, respectively. There was no difference in the conception rate between the two protocols. Conception rate after artificial insemination (AI) or embryo transfer (ET) were 46.3% and 51.4%, respectively. The risk factors affecting conception rate in heifers were daily weight gain (odds ratio [OR]= 4.673; P= 0.036) and body condition score (BCS) (OR= 3.642; P= 0.018). Furthermore, estrus synchronization protocol (OR= 1.774; P= 0.083) and the absence of corpus luteum (CL) at the initiation of treatment (OR= 0.512; P= 0.061) had a tendency to affect the conception rate, while age (OR= 0.715; P= 0.008) was a protective factor to conception rate. In conclusion, positive daily weight gain before AI or ET, higher BCS, younger age, and the presence of CL at the initiation of estrus synchronization in dairy heifers increased the likelihood to conceive.


INTRODUCTION
One of the reasons in declining fertility in dairy cows is the increased capability for milk production (Butler, 2003). The detrimental effects of negative energy balance (NEB) in early lactation appear to be manifested as the problem during the breeding period, resulting in a low fertility. In dairy heifers, a better fertility than in lactating dairy cows has been reported (Pryce et al., 2004). This difference may be due to the difference in the metabolic and physiological status between cows and heifers (Royal et al., 2000). The more dairy cows lose body condition score (BCS) during the postpartum period, the lower is their conception rate. Cows losing one unit or more BCS (five-point scale) during early lactation are at a greater risk for low fertility. In cows, nutritional requirements increase rapidly with milk production following parturition, and the resulting NEB extends for 10-12 weeks, that is strongly associated with the length of the postpartum unovulatory period (Butler, 2003).
A number of factors influence conception rate (CR) in heifers after artificial insemination (AI). Change in body weight and/or BCS before breeding could be one of the important factors affecting CR in heifers. Brickell et al. (2009) reported that heifers with faster growth rates had a lower age at first breeding as well as a lower age at first calving. It has also been believed that heifers gaining body weight show a higher CR after embryo transfer (ET) than those losing weight. We assumed that heifers gaining weight before AI or ET after induction of estrus have a higher conception rate than those not gaining weight.
This study aimed to show the risk factors affecting the conception rate in Holstein heifers after synchronization of estrus.

Animals and Housing
Holstein-Friesian heifers which were being reared at Yamaguchi Prefectural Heifer Raising/Breeding Farm, Yamaguchi, the south-western region of Japan, were used for the experiments. The calves at the age of 1 to 3 months were brought from a number of commercial dairy farms in Yamaguchi Prefecture to the farm and kept there until they became pregnant after AI or ET under the contract. Those pregnant heifers were returned to each farm a few months before due date.
The experimental heifers were housed in a free barn where the concrete floor was covered with sawdust. They were fed a diet formulated in line with NRC recommendation.

Reproductive Management
Estrus was detected by visual observation as a routine twice a day (09:00-10:00 and 15:00-16:00 h) with the help of tail paint (ALL-WEATHER ® PAINSTICK ® , LA-CO Industries Inc., Elk Grove Village, IL, USA). Heifers which were in estrus at 09:00-10:00 or 15:00-16:00 were inseminated artificially by veterinarians within one hour after the confirmation of estrus. Embryo transfer (ET) was conducted 7 days after estrus by the veterinary staff.
The heifers were bred either by AI or ET according to the request of the owners. The owners generally preferred for their heifers to be transferred with embryos from Japanese beef cattle for the first breeding.
In case the heifers did not conceive after ET, the owners requested the farm to do AI for subsequent breeding. Pregnancy was diagnosed by palpation per rectum at 35 d or more after AI or 28 d after ET or later.
Those heifers which had not shown signs of estrus for more than one month were referred to the experiment. In addition, the heifers which had been subjected to ET 7 d after estrus, but were not suitable for ET due mainly to the formation of poor CL were also used for the study.

Data Collection for Factors which may Affect Conception Rate after AI or ET
The effects of eight factors on conception rate in heifers were investigated in this study; protocol for synchronization of estrus, breeding methods, stage of estrus cycle at the initiation of treatment, body condition score (BCS), body weight, daily weight gain, changes of body weight, and age (Table 1).

Protocols for Synchronization of Estrus
The herd was visited regularly at 4 week intervals for synchronization of estrus. A group of 4 to 14 animals, depending on the availability, were referred to the experiment at each visit. During a period from August 2005 to August 2008, the animals were allocated into 2 treated groups (Heatsynch and CIDR-Heatsynch groups) without regarding to the stages of the estrus cycle at the beginning of treatment (Yusuf et al., 2010;Bridges & Lake, 2011). During the following period from September 2008 to November 2009 all animals were referred to only CIDR-Heatsynch protocol.
In CIDR-Heatsynch group (n= 176), heifers were injected with GnRH, PGF 2α , and EB, similar to heifers in Heatsynch group. However, in addition, a CIDR (EAZI-BREED CIDR ® (Livestock Improvement Association of Japan, Tokyo, Japan) was inserted into the vagina at the time of GnRH injection and was removed on the day of PGF 2α injection (Yusuf et al., 2010). All heifers were applied with heat detection devices; KAMAR ® heat mount detector (HMD) or ALL-WEATHER ® PAINTSTIK ® (tail paint). The changes of the heat detection devices were monitored twice daily. The animals showing positive response of the devices, wearing off the tail paint of 50% or more, and the reac-tion of HMD with red color were palpated per rectum.
Heifers showing clear uterine contraction were confirmed to have been in estrus (Yusuf et al., 2010).

AI or ET
Heifers assigned to AI were inseminated artificially with frozen/thawed semen from proven sires within 1 h after confirming estrus. For ET, a fresh embryo of grade A or B or grade A of frozen/thawed embryo was nonsurgically transferred into the uterine horn ipsilateral to the corpus luteum 7 d after the estrus was detected.

The Presence of CL at the Initiation of Treatment
Synchronization of estrus in both protocols was initiated at random stages of the estrus cycle. Before administration of GnRH, all heifers were palpated per rectum to examine the ovarian structures. Heifers with inactive ovaries were excluded from the study. The animals were classified into those with corpus luteum (CL) and the others without CL based on palpation per rectum as well as plasma progesterone determination. Blood samples were collected via coccygeal venipuncture into evacuated heparinized vacuum tubes (VENOJECT ® II TERUMO, Terumo Corporation, Tokyo, Japan) for determination of plasma progesterone concentrations at the initiation of protocol. The samples were centrifuged at 1500 x g for 15 min immediately after collection. The plasma was collected and stored frozen at -20 o C until the analysis of progesterone concentrations by enzyme immunoassay (EIA) validated by Isobe & Nakao (2003). Intra-and inter-assay coefficients of variation were 5.7% and 6.6% in the plasma with 11.8 ng/mL, 19.1% and 19.3% in the plasma with 0.5 ng/mL, respectively.
All heifers with CL showed 1.0 ng/mL or higher plasma progesterone concentrations. Among the heifers with CL or dominant follicle (DF) which had not been classified by palpation per rectum, those with progesterone concentrations of 1.0 ng/mL or higher were grouped to have CL, while those with the progesterone concentrationss less than 1.0 ng/mL were grouped to have DF.

Body Condition Score
Heifers' body condition score was determined at the beginning of the experiment by the same person throughout the experiment using a-5 point system (1= thin to 5= fat) in increments of 0.25 (Edmonson et al., 1989).

Body Weight
Body weight of heifers was measured routinely by a weight measurement scale at one month interval to monitor their growth. Heifers reaching body weight of 350 kg and age of 12 months were submitted to the first breeding. Daily weight gain in the present study was calculated by the change in body weight during a period of one month before AI or ET including a period of synchronization treatment divided by the days.

Statistical Analyses
Statistical package SPSS 12.0 for windows (SPSS Inc., Chicago, Illinois, USA) was used in the study. AI submission rate and ET submission rate were calculated as the number of heifers inseminated or transferred with an embryo divided by the number of heifers to be inseminated or to be transferred with an embryo. Conception rate was calculated as the number of heifers conceiving divided by the total number of heifers inseminated or embryo-transferred. The pregnancy rate was defined as the number of heifers conceiving divided by the total number of heifers to be inseminated or to receive an embryo transfer. The percentages of heifers coming into estrus before PGF 2α treatment between Heatsynch and CIDR-Heatsynch groups were compared using Fisher's exact test, whereas the percentages of heifers coming into estrus within 5 d after PGF 2α treatment between the 2 groups were compared using Chi-square test. Chi-square was also used to compare the combined percentages of heifers coming into estrus before and within 5 d after PGF 2α treatment, AI or ET submission rate, conception rate, and pregnancy rate after AI or ET between Heatsynch and CIDR-Heatsynch groups.
The binary logistic regression model was used to analyze the effects of body weight change before AI or ET and the other possible factors affecting the reduced likelihood of heifers to conceive. All calculations were categorized having significant effect if the P-value is less than 0.05.

Reproductive Performance in Heifers after Induced Estrus
Reproductive performance in dairy heifers after induced estrus using Heatsynch and CIDR-Heatsynch is shown in Table 2. The overall estrus induction rate was 94.5%. In Heatsynch group, 17 heifers (17.2%) came into estrus before PGF 2α treatment, but none in CIDR-Heatsynch group.
The overall conception rate in the present study, including those with different treatment protocols and those with different breeding methods was 48.2%.
AI and ET submission rates, conception rate, and pregnancy rate did not differ between Heatsynch and CIDR-Heatsynch groups ( Table 2). The conception rate at different variables and classes are shown in Table 3. Table 4 presents the binary logistic regression model for the factors causing the failure of heifers to conceive. Daily weight gain and BCS were the factors which contributed to the failure of heifers to conceive. Protocol and the presence of CL at the initiation of treatment had a tendency to affect the conception rate. As the BCS of the heifers increased by 0.25 unit, the likelihood of heifers to conceive increased by a factor of 3.624. Similarly, for the increased daily gain, the chance for heifers to become pregnant increased by a factor of 4.673. Age was a protective factor to conception rate, the increased age of animals affected the probability to conceive decreased by a factor of 0.715.

DISCUSSION
In this study, we analyzed the effects of daily weight gain before AI or ET and other factors on conception rate in heifers. The factors include the protocol for synchronization of estrus, breeding method, the presence of CL at initiation of treatment, BCS, body weight, and age. The lower BCS of the heifers at the initiation of treatment, negative daily weight gain, and the increased age of animals were the risk factors to reduce the probability of the heifers to become pregnant. Heifers with CL at the initiation of treatment had more chance to conceive than those without CL.
The occurrence of estrus before PGF 2α treatment in Heatsynch group might be caused by the different stages of the estrus cycle in the initiation of protocol, Note: a,b Means in the same row with different superscripts differ significantly (P<0.01).   (2011) and Thomas et al. (2016) using beef heifers. This result is related to the formation of CL with normal life-span following CIDR removal (Wheaton & Lamb 2007), the suppressed estrus during CIDR insertion that subsequently allow a high rate of submission for AI (Rabaglino et al., 2010;Palomares et al., 2015;. The conception rate after AI at inducing estrus or ET 7 d after the estrus obtained in the present study was compared with the results of previous studies using some combinations of hormonal treatment; GnRH, PGF 2α , EB, estradiol cypionate (ECP), and intravaginal device (CIDR) (Cavalieri et al., 2007;Silva et al., 2015;Pereira et al., 2016;Say et al., 2016).
Estrus detection rate within 5 d after PGF 2α treatment in Heatsynch group was 76.8%. When the heifers detected in estrus before PGF 2α injection were included, the total estrus detection rate within 12 d after the initiation of the protocol was 93.9%, equivalent to the estrus detection rate in the animals treated with CIDR-Heatsynch. The high estrus induction rate achieved in the present study might be due to the use of heat detection devices, HMD or TP, which were effective to improve the estrus detection rate. Although the estrus induction rate was high in the present study, the pregnancy rate was lower than the rate at spontaneous estrus as reported in previous studies (Cavalieri et al., 2007). Likewise, the study of Dorsey et al. (2011) showed that pregnancy rates to AI were significantly affected by the time of insemination relative to the onset of estrus.
The higher BCS of the heifers in the present study resulted in the higher conception rate after AI or ET. Similar result was obtained in beef cattle using fixed time AI protocols (Richardson et al., 2016). Low conception rate in heifers with low BCS might not be due to anovular conditions found in lactating dairy cows (Chebel et al., 2006), since all the heifers were confirmed to have ovulated one day after estrus.  (Chebel et al., 2007) ᵇ Increased >0.05 kg/day; stable = -0.10-0.05 kg/day; decreased <-0.10 kg/ day. In this study, we emphasized how daily weight gain affects conception rate in heifers in comparison to the other factors. Out of eight factors included in the present study, daily weight gain was the third factor affecting conception rate in heifers after age and BCS (Table 4) and followed by the presence of CL at the initiation of treatment and estrus synchronization protocol. Heifers with moderate daily weight gain at around the initiation of the study had higher conception rate than thin or heavy heifers. This result is consistent with the study of Chebel et al. (2007). The body weight of heifers did not affect conception rate after AI or ET (Table 3).
Changes in body weight before treatment as was shown by the daily weight gain had an influence on conception rate (Table 3). Heifers gaining more body weight had more chance to conceive than those heifers gaining less body weight. This results was consistent with the study of Brickell et al. (2009). Their study showed that poor growth with low circulating concentrations of IGF-1 during the rearing period was the main factor associated with the delayed first breeding and first calving.
The age of the heifers was found to affect the conception rate in the current study. This result was similar to that reported in the study of Kuhn et al. (2006). They demonstrated that the age heifer was the second most important main effect in terms of magnitude of impact on the conception rate. Furthermore, they reported that breeding heifers at the age of 15 to 16 months maximized the conception rate, and the conception rate was lower either for breeding at the age of <15 months or for breeding at the age of >16 months. In the present study, heifers bred at the age of less than 14 months showed a relatively higher conception rate than those bred at the age of greater than 14 months. This implied that heifers bred at the age of greater than 14 months required more inseminations to conceive. The addition of CIDR in the protocol prevented the occurrence of premature estrus and resulted in a higher synchrony of estrus after treatment and formation of a CL with normal life-span (Wheaton & Lamb, 2007). Similar results were reported by Bishop et al. (2016) using the 14-d CIDR-PGF 2α protocol. Their study used pubertal and prepubertal or peripubertal heifers in comparison to lactating cows. This result indicated that inclusion of CIDR in a protocol suppressed estrus during CIDR insertion. Likewise, the use of long-term CIDR insert-based protocol (Leitman et al., 2009) using beef heifers resulted in a high rate of estrus response.
Stage of estrus cycle at the initiation of the treatment affected the conception rate. The conception rate in heifers with CL at the initiation of protocol tended to be higher than those in heifers without CL. Contrary, low peripheral progesterone concentrations during follicular development may lead to the decreased fertility (Atanasov et al., 2015). The presence of CL at the initiation of estrus synchronization increased the likelihood of heifers to conceive. This might probably due to fact that heifers that were in luteal phase at the beginning of protocol showed clear estrus signs than heifers that were in the follicular phase. The presence of CL at the initiation of estrus synchronization coincident with the administration of GnRH, most probably affected the oc-currence of ovulation (Imron et al., 2015), the emergence of a new follicular wave that subsequently affect the expression of estrus (Sahu et al., 2014). The expression of estrus in cattle after synchronized estrus is associated with the increased fertility (Pereira et al., 2016).

CONCLUSION
Positive daily weight gain before AI or ET, higher BCS, younger age, and the presence of CL at the initiation of estrus synchronization in dairy heifers increased the likelihood to conceive.