Pregnancy Rate and Reproductive Disorders Examination of Inseminated Brahman Cross Cows by Rectal Palpation and Ultrasonography

Corresponding Author: Mohamad Ervandi Faculty of Agricultural Science, Muhammadiyah University of Gorontalo, Gorontalo 96181, Indonesia Email: ervandi_husain@yahoo.co.id Abstract: Pregnancy is a condition when a fetus began to develop in the uterus of the cow. Early pregnancy detection becomes important as it would determine the success of breeding management as well as to achieve the best productivity. This study aimed to evaluate and observe the pregnancy rate and reproductive disorders of Brahman Cross (BX) cows and compare the results to the Ultrasonography (USG) observation. The materials of the study were 80 BX cows selected through a purposive sampling method with the following criteria: aged 2-3 years, weighed 300-500 kg, has at least calved once and had the body condition score (BCS) at 4-7 (in 1-9 scale). This study was conducted as experimental research, with 40 BX cows were artificially inseminated with Ongole crossbred (PO) semen at 0-2 h interval and the other 40 BX cows were at 8-10 h interval from the estrous detection. All of the BX cows were firstly synchronized with PGF2α (LUTALYSE) and then inseminated with PO semen (>40% motility) as according to the Indonesian standard. The pregnancy rate of the BX cows was observed through rectal palpation and 17β-estradiol hormone observation and further examined for the reproductive disorders by using USG. The obtained data were then analyzed descriptively and the results showed that 0-2 h insemination had a higher success of pregnancy (32.5%) compared to the 8-10 h interval (27.5%). Moreover, the highest 17βestradiol in the inseminated BX cows was at 5,765 ng/ml, while the lowest was at is 0.41 ng/ml. The USG observation showed that 6 BX cows (7%) in this study had a follicular cyst, 2 BX cows (2.25%) had persistent corpus luteum and 1 BX cows (1.25%) had ovarian hypofunction.


Introduction
Beef cattle have been the main meat producers and source of the nutritional animal-based protein. The demand for beef consumption in Indonesia has been increasing along with the increased human population and could not be fulfilled solely from the local breeds. The breeding of local cattle with other breeds with higher meat production provides an alternative way to fulfill the demand for meat consumption. Brahman Cross (BX) cattle is one of the crossbreed cattle developed in Indonesia aimed to achieve the goal. The BX cattle had shown good adaptability to the tropical climates, survivability to ectoparasites, as well as good body weight gain and productivity even fed with a low quality feed. The production performance of BX cattle, thus showed its potential to be reared in Indonesia in order to increase domestic meat production (Kuswati and Susilawati, 2016).
The common breeding practice in Indonesia is by natural breeding or Artificial Insemination (AI). The AI has shown many beneficiaries compared to the natural breeding, however, the success of AI would be determined by several factors such as heat detection, inseminating skills and the body condition of the cows (Saacke, 2008;Roelofs et al., 2010;Susilawati, 2011). Silent heat is commonly cases found on the farm which will prevent farmers to make the correct decision to determine the time for artificial insemination. Yekti et al. (2017) described that silent heat is the condition where cows did not express clear signs of heat due to the low estrogen level.
Pregnancy is defined as the post-mating physiological period that resulted in the conception and then followed with embryo development into a fetus until parturition (Hafez and Hafez, 2000). Various pregnancy diagnosis can be done depending on the reproductive anatomy and physiological condition of the livestock. In large livestock, such as cows, the most practical and reliable diagnosis is by detecting rectal palpation or by using Ultrasonography (USG) (Fricke, 2005).
The current conditions showed that the AI of BX cows still had the low success of pregnancy. Research by Annashru et al. (2017) showed that different time of AI would affect the calving to non-return rate of BX cows, with 0-4 h insemination interval from detected estrous was higher (70%) compared to the 8-12 h (37.14%). The condition thus showed that the time interval of insemination could affect the success of AI as well. In this study, we observe the different time intervals of insemination to the pregnancy rate and occurred reproductive disorders in BX cows.

Materials
The materials of this study were 80 Brahman Cross (BX) cows selected through a purposive sampling method with the following criteria: aged 2-3 years, weighed 300-500 kg, have at least calved once and had the body condition score (BCS) at 4-7 (in 1-9 scale).

Methods
This study was conducted as experimental research. All of the BX cows were firstly synchronized with PGF2α (LUTALYSE) and then inseminated with PO semen (>40% motility) as according to the Indonesia standard. The first treatment (P1) were 40 BX cows inseminated at 0-2 h intervals from estrous detection, while the second treatment (P2) were 40 BX cows inseminated at 8-10 h intervals from estrous detection. The pregnancy rate of the BX cows was observed through rectal palpation at 90-days after insemination, while the 17β-estradiol hormone measurement was done by firstly taking the blood sample of BX cows from the jugular vein in the tail area at 90-days after insemination. The sample was then measured for the 17β-estradiol hormone by using commercial ELISA kit (Bioassay Tecnology Laboratory, Shanghai) according to the manufacturer's instruction: http://www.btlaboratory.com/product/bovine-estrogen-elisa-kit-2/. The USG (Mindray DP-50) observation was done by following Royal et al. (2000). The pregnancy rate was analyzed descriptively and formulated as follows:

The Pregnancy Rate of the Brahman Cross Cows
The pregnancy observation by using rectal palpation of BX cows in this study is presented in Table 1.
The results showed that the overall successful pregnancy of artificially inseminated BX cows both at P1 and P2 was 30%. Furthermore, the pregnancy rate at P1 or 0-2 h insemination interval (32.5%) was higher compared to the P2 or 8-10 h interval (27.5%). The low successful pregnancy of the BX cows was caused by disorders in the ovary, especially ovarian hypofunction, follicular cyst, persistent corpus luteum and endometritis ( Fig. 1 to 3).
The rectal palpation showed that 56 out of 80 inseminated BX cows were failed to pregnant. Purposive sampling was done by selecting 18 BX cows, consisted of 6 BX cows with positive (+) NRR and rectal palpation; 6 BX cows with positive (+) NRR but negative (-) rectal palpation; and 6 BX cows with negative (-) NRR and rectal palpation, for USG examination. The estradiol 17-β levels of each cow were measured to understand the relationship of the hormone concentration to the status of pregnancy of the BX cows' ovary (Table 2).

Ultrasonography Examination
It can be seen that the highest 17β-estradiol concentration levels of the BX cows were 5,765 ng/ml while the lowest was 0.41 ng/ml, with each BX cow had varying 17β-estradiol concentration levels. The observed 17β-estradiol concentration levels in this study are higher than the finding by Domènech et al. (2011) in dairy cows, which showed 17β-estradiol concentration levels at 26.75±8.63 pg/ml or equals to 2,675 ng/ml; and Kajaysri (2006) which showed the 17β-estradiol concentration levels in Friesian crossbreed dairy cows were 2,786 ng/ml.

Ultrasonography Observation
Physiological observation of the BX cows' ovary by using ultrasonography showed that 6 cows (7%) had a follicular cyst, 2 cows (2.25%) had persistent corpus luteum and 1 cow (1.25%) had ovarian hypofunction.    Description: The non-return rate observations were performed at 18-days (NRR 1 ), 36-days (NRR 2 ) and 54-days (NRR 3 ) after insemination. The rectal palpation and ultrasonography examinations were performed 90-days after insemination.   In Fig. 1, it can be seen that the BX cows had ovarian hypofunction, characterized by the slippery ovarian surface during rectal palpation due to the undeveloped follicles and corpus luteum. It is allegedly caused by the feed deficiency of the BX cows which resulted in impaired FSH and LH secretion. Endometritis and persistent corpus luteum with 2.59 mm in length and 1.58 mm in width were also found in the BX cows in this 2a 2b 3a 3b study (Fig. 2). The USG observation with normal ovary and 4-months fetus can be seen in Fig. 3.

Pregnancy rate of the Brahman Cross cows
In this study, energy deficiency from the feed is known to contribute to the low rate of successful pregnancy, noting that it would affect the FSH and LH hormone secretion, thus disrupt ovulation, fertilization and embryonic development as well as causing ovarian hypofunction. Boland and Lonergan (2003) reported that nutrient deficiencies can reduce fertility, whereas Yekti et al. (2017) added that low feed quality and quantity will cause sub-fertilization, especially in exotic crossbreed cows. Zare et al. (2008) showed the impact of improved nutrition would initiate the hypothalamus to affect the anterior pituitary gland to release gonadotropins and growth hormones, thus affect ovulation followed by increased estradiol l7-β levels. Another known factor that contributes to the successful pregnancy is the proper heat detection of the inseminated cows. The heat occurrence on the acceptor is affected by gonadotropin and FSH hormone induction, which stimulates the development of primary and de Graff follicles, especially granulosa cells which, then produce the 17-β estradiol and LH hormone and stimulates ovulation (Hafez and Hafez, 2000).

Relationship between Estradiol 17-β Concentration and Ultrasonography Examination
A large follicular cyst was found in the BX cows' ovary ( Fig. 1), which then caused nymphomanic condition (continuous heat) and affect the 17β-estradiol levels. This is in accordance with Nora et al. (2018), who showed that large follicles, as well as the occurrence of the follicular cyst, would result in high 17β-estradiol levels. The occurrence of a follicular cyst could be detected if the follicle is larger than 25 mm without any corpus luteum whether on both left or right ovary (Murayama et al., 2015).
The blood sample collection in this study was carried out at 9 AM before the ultrasonography (USG examination. The time for blood collection is essential as the cows would have a varying physiological condition, thus affect its 17β-estradiol levels. Hafez and Hafez (2000) mentioned that during a oestrous cycle, reproductive hormones interact with each other and affect its physiological and behavioral changes of the cow. Hossner (2005) added that hormones are influenced by internal and external factors. The internal factors include the genetic and physiological conditions, while the external factors include nutrition intake and the environment condition.
The ultrasonography observation showed that 6 cows had follicular cysts, 2 cows had persistent corpus luteum and one cow had ovarian hypofunction. The follicular cyst would cause reproductive failure and mostly found with the diameters at more than 2.5 cm, attached in the ovary for 10 days or more, without any presence of corpus luteum. The follicular cyst is known to disrupt ovulation and decreased LH hormone production. Hermadi et al. (2011) explained that follicular cyst will occur due to the lack of LH hormone production, while the FSH hormone remained sufficient, thus stimulate the production of the anovulatory follicle and resulted in nymphomanic condition of the cow. Yekti et al. (2017) added that nymphomania in cow indicates the occurrence of follicular cysts.
Moreover, endometritis and persistent corpus luteum were also found in the BX cows in this study (Fig. 2). This condition was caused by whether infection or high progesterone levels suppress the FSH and LH secretion from the anterior pituitary gland. Research by Ghanem et al. (2015) has elucidate various bacterial infection to the occurrence of endometritis, while Sheldon et al. (2006) showed that high progesterone level in blood contributes to the persistent corpus luteum.
The ovarian hypofunction occurs due to the low FSH and LH hormones production and resulted in the failure of follicle production in the ovary. Hermadi et al. (2011) stated that the bad physiological condition, as well as long feed deficiency of the cow, would alter the ovarian hypofunction into ovarian atrophy. Moreover, the persistent corpus luteum would occur due to the failed luteolysis and the corpus luteum remained in the ovary in a long time despite the failed pregnancy. All form of the corpus luteum can produce the progesterone hormone, this explains that the BX cows with persistent corpus luteum in this study had high progesterone hormone levels in the blood. Boland and Lonergan (2003) mentioned that nutrition deficiency would reduce the cows' fertility and research by Zare et al. (2008) showed that, the improved nutritional intake would affect the anterior pituitary gland to produce higher gonadotropin and growth hormone, thus resulted in higher ovulation and 17β-estradiol levels. In addition, Feradis (2014) showed that nutrition deficiency would disrupt the gonadotropin synthesis in the pituitary gland.

Relationship between Non-Return Rate, Estradiol 17-β Concentration and Ultrasonography Examination
In this study, pregnancy examination was done by observing the NRR, rectal palpation and further confirmed by ultrasonography examination. The 17βestradiol levels were also measured to understand its relationship to the status of pregnancy. The ultrasonography observation of the BX cows showed various ovarian abnormalities, such as persistent corpus luteum, ovarian hypofunction and follicular cyst. Hafez and Hafez (2000) described that the ovarian hypofunction is caused by the failure of follicular cells to respond to the hormonal stimulation, changes in hormonal secretion, decreased stimulation in the ovarian hypothalamic-pituitary function which resulted in no ovarian activity after calving. The clinical symptoms of follicular cyst include nymphomania (continuous heat), anestrus (silent heat), sacroischiadicum ligament relaxation, vulvar edema and increased uterus size. Noakes et al. (2016) showed that follicular cyst with diameters at 1-3 cm can be felt when touched using rectal palpation. The occurrence of a follicular cyst would cause ovulation failure. Ovulation requires a series of processes at the cellular and subcellular levels involving hormones and factors in the form of complex biochemical compounds (Russell and Robker, 2007). The existence of metabolic infertility is closely related to malnutrition which will lead to the low reproductive performance of cows (Donaldson, 2014). In addition, Dibia et al. (2015) mentioned that another factor that needs to be further observed is the high cases of ovarian hypofunction in productive cows due to crossbreeding.
The occurrence of persistent corpus luteum (Fig. 2a) and endometritis (Fig. 2b) indicates that the BX cows suffer pyometra. Endometritis is caused by the failed PGF2α production in the endometrium, which then resulted in the failed luteolysis. Santoso et al. (2014) described that persistent corpus luteum often found in cows with reproductive disorders, indicated with continuous progesterone production and longer heat cycle. However, in some cases, cows could still get pregnant, but the fetus would still die to the failure involution (Sayuti et al., 2012;Cuneo et al., 2006). Siregar et al. (2012) described that small follicles (3-8) with a total of fewer than 10 follicles are categorized as dominant follicles, while small follicles with a total of more than 10 follicles were not. This study showed that the examination of dominant follicles is based on its morphology, the results of the study indicate that follicular growth is a dynamic process controlled by local and systematic processes (Fortune et al., 2004). Large follicles that appear during luteolysis will become dominant and subsequently ovulate in the follicular phase (Inskeep, 2004).
Pregnancy is a period from fertilization until giving birth (Frandson et al., 2009). The risk of failed pregnancy would be higher during early pregnancy, specifically at day-2 until day-42 after insemination. The early ultrasonography examination would produce more accurate failed pregnancy detection compared to rectal palpation (De-Vries, 2006). Moreover, feed also known to play important roles in the reproduction process.
Research by Susilawati (2013) showed that feed would affect all cows' activities, starting from the cows' metabolism, growth, lactation and also reproductive activities. Therefore, special concern in nutritional intakes of the cows should be taken, especially in the proteins, carbohydrates, vitamins and mineral intakes, as the deficiency of the compounds would cause continuous reproductive disorders.

Conclusion
This study concludes that the 0-2 h artificial insemination interval after estrous detection of BX cows had a higher pregnancy rate (32.5%) compared to 8-10 h interval (27.5%). Moreover, the 17β-estradiol measurement showed that the highest 17β-estradiol levels of BX cows in this study were at 5,765 ng/ml, while the lowest at 0.41 ng/ml. The ultrasonography observation showed that 6 out of 80 BX cows (7%) had ovary abnormalities due to the occurrence of the follicular cyst, 2 cows (2.25%) had persistent corpus luteum and 1 cow (1.25%) had ovarian hypofunction.