SUCCESS OF PROTOCOLS FOR SYNCHRONIZATION OF ESTRUS AND OVULATION IN DAIRY COWS IN SUMMER

Cattle farming, as one of the most important branches of livestock production, has long been confronted with the chronic problem of the decline in reproductive performances. In 2005, the number of cattle around the world amounted to about 1,370,000,000 heads of cattle, while in 2012 this number was just over a billion, which indicates the importance of applying different reproductive protocols in order to increase production. Heat stress leads to disorders of the physiological and reproductive processes, as the rise in body temperature caused by heat stress has direct negative consequences on numerous cell functions. The study included a total of 54 HolsteinFriesian cows, 28 experimental and 26 control cows from PD Butmir farm with 5 lactations on average. Hormone protocols for estrus and ovulation were used in June and July 2013. Cows in the experimental group were subjected to a Presynch + 5dCoS2 protocol. Cows that did not conceive after this protocol were immediately subjected to resynchronization with Cosynch -72 protocol. The success rate of the Presynch + 5dCoS2 protocol was 19%, while in the case of Cosynch 72 it was 33%, which was statistically significant (p <0,05). The first presynchronization and synchronization protocol (5dCoS2) did not significantly help to improve the conception rate after the first insemination postpartum, but it is evident that they had a positive effect on another protocol (COS72) in the form of reduction in embryonic mortality in the summer months when embryonic loss is the greatest. The COS72 protocol provides satisfactory results in the summer, but good fitness and health management, as well as heat stress reduction in accordance with the location and farm design are the preconditions for a successful estrus and ovulation synchronization program prior to artificial insemination.


INTRODUCTION
Cattle farming, as one of the most important branches of livestock production, has long been confronted with the chronic problem of the decline in reproductive performance.In 2005, the number of cattle around the world amounted to be approximately 1,370,000,000, while in 2012 this number was just over a billion, which indicates the importance of applying different reproductive protocols in order to increase production.For successful reproduction, several prerequisites are needed such as the successful development of oocytes in the follicles, the expression of oestrous behavior and coordinated ovulation, the optimum uterine lining for the development of embryos and effective reproductive management.The age of cattle is also very important for reproductive efficacy.At the age of 700-750 days, primiparous cows have 118-days service period, first artificial insemination after 66 days, conception rate after the first insemination of 36.4% and an artificial insemination index of 2.85 (Ettema et al., 2004).Fertility begins to decline with age, compared to older cows, primiparous cows have a higher conception rate after the first insemination postpartum, while cows with ≥5 lactations have the lowest conception rate (Pursley et al., 1993).Heritability of positive reproductive parameters is ≤ 3% (Hansen, 2000).Holstein-Friesian breed has a lower success rate of the first insemination postpartum than other breeds of dairy cows and cross-breed cows, as well as a greater inclination to metabolic disorders (Pursley et al., 1993, Hansen, 2000).Reproduction is more efficient in cross-breed cows because they have a better conception rate after the first insemination postpartum (51-56% cross-breed cows, 40% Holstein breed) and less embryonic deaths (7.5-10.1% cross-breed cows, 12.8% Holstein breed) (Rabiee et al., 2005).A higher metabolic and nutritive load due to higher milk production can be responsible for the decline in reproductive performance, as Holstein Friesian cattle produce the highest amount of milk with the highest fat and protein content, but they also have the shortest productive lifetime compared to other dairy cattle, for example Norwegian Red cattle breed (with the longest lifetime production) (Walch et al., 2008).High milk production may have an antagonistic effect on estrus expression, but not on the reactivation of ovarian function (Harrison et al., 1990) High-producing cows have the longest interval to the first ovulation and estrus with low intensity of estrus expression, while other authors claim that highproducing cows, due to better general health status and multiple double ovulation, have greater chances of conception (Peters andPursley, 2002, Santos et al., 2004).Due to increased catabolism higher milk yield is associated with reduced reproductive performance and delayed ovulation but effective management can solve this problem (Santschi et al., 2011).At higher ambient temperatures and heat stress, follicle growth is threatened, and hormone concentrations associated with earlier cycle ( Reist et al, 2003) are also reduced.Heat stress can disrupt physiological and reproductive processes (Hansen and Arechiga, 1999).The rise in body temperature caused by heat stress has direct negative consequences on cell functions.At higher temperatures and heat stress, follicle growth is endangered and the concentrations of progesterone, triiodothyronine and thyroxine (E2, T3, T4) are reduced (Reist et al., 2003, Sota et al., 1998).Heat stress in the summer reduces the length and intensity of estrus and therefore there is 76 -82% of undetected estrus events and 44-65% of them in winter.(Pursley et al., 1998).Heat stress can also cause increased cortisol secretion (ACTH response) which blocks estradiol-induced sexual behavior (Pursley et al., 1998).Stress caused by elevated air temperature reduces libido, fertility, increases body temperature and embryonic mortality (Santos et al., 2009).Negative effects of heat  (Jordan, 2003).Heat stress occurs when the air temperatures are> 20 ° C, while the upper critical temperature at the start of the heat stress is between 25-26 ° C (Alkatanani et al, 1999).Increased temperature and humidity reduce appetite and dry matter intake, expression and duration of estrus, colostrum quality, endocrine status, conception rate, early embryonic development and fetal growth and the interval partus-conception is prolonged to extend the state of the negative energy balance (NEB) (Pursley et al., 1998, Jordan, 2003, Hansen and Arechiga, 1999).Heat stress affects oocyte maturation in the dominant follicles (Wolfenson et al., 1997), resulting in less viable embryos.In warm months (May-August) when air temperature and humidity is high, Bos taurus cows have lower oocyte quality (41 ± 9.5%) as well as lower embryo quality compared to the colder period of the year (75.9 ± 8%) (January-March) (Rocha et al., 1998).When it comes to reproduction hyperthermia is the most harmful on the day of estrus or the day after (Sota et al., 1998).Redirection of blood from the core to the periphery which results in reduced uterine and placental perfusion pressure is the adaptive response to heat stress (Hansen and Arechiga, 1999).Poor blood flow and tissue perfusion, disturbed endometrial and oviduct function and concentrations of steroid hormones enable secretion of prostaglandin F2α causing embryonic mortality (Pursley et al., 1998, Xu andBurton, 2000).Dry matter intake and milk yield is often stimulated by increased protein intake, but this nutritional strategy also leads to reduced fertility, as excess of rumen degradable and rumen undegradable protein can contribute to reduced fertility in lactating cows (Butler, 1998).In order to stimulate milk yield, meals have> 17% of crude protein (SP) resulting in reduced reproductive performance.Excess protein in high-producing cows reduces reproductive efficiency and it doesn't increase milk yield.In NEB cases there is a lack of energy for detoxification of ammonia and there are changes in urea concentrations in blood and milk which are greater than 19 mg/ dl and can be harmful to conception and embryonic evelopment.Endometrial cells respond to this by intensifying the secretion of prostaglandis, decreasing progesterone concentrations and embryo survival rate (Butler, 1998).Body condition score (BCS) reflects the body energy reserves available for metabolism, growth, lactation and ovarian activity (Montiel and Ahuja, 2005).This together with the presence of lameness speak about the welfare of animals on the farm, which is important for reproductive and productive performances.The occurrence and duration of NEB is associated with the interval to the first ovulation PP, and it is assumed that NEB in early lactation adversely affects the quality of the oocyte in 80 to 100 days period, reducing the conception in the first few inseminations (Patton et al., 2006).Adipose tissue constitutes the major β-carotene storage which affects the ovulation, luteal function, and progesterone levels (P4) and a loss in body condition results in decreased levels of βcarotene in plasma (Kawashima et al., 2009).

MATERIAL AND METHODS
The study included a total of 54 cows of Holstein-Friesian breed, 28 experimental and 26 control cows from PD Butmir farm (Canton Sarajevo).Average annual milk yield per cow on this farm is around 6,000 litres and 5 lactations.Estrus detection is carried out three times a day, visually on the basis of external signs and, if necessary using vaginal temperature measurements and transrectal examination.The voluntary waiting period for this farm is 60 days, followed by a clinical examination of the ovaries and uterus.After the examination it is decided The following reproductive parameters were analyzed: the interval to the first postpartum insemination, the success of the conception after the first and subsequent postpartum inseminations, insemination rate, the length of the service period, and the total number of pregnant and non-pregnant cows.

Ultrasound Measurements for Body Condition Score (BCS)
In addition to the first artificial postpartum insemination, body condition status in the experimental and control group was determined by sonography based on the thickness of the subcutaneous fat tissue.It is a fast, non-invasive method in which the ultrasonic probe is placed in the area of the sacrum and the root of the tail, because the largest deposits of adipose tissue are on the back.Hair doesn't need to be cut for the examination and a plant oil is used as a contact agent between the probe and the skin.The probe is positioned vertically and it must be held lightly because fat is compressed with pressure.The adipose tissue is seen as a dark (hypoechoic) narrower or thicker line, while the skin and profound fascia are light (hyperechoic).The thickness of the skin is always calculated, and the thickness of the subcutaneous adipose tissue is expressed in millimeters.
Figures 1 and 2. Location for placing an ultrasound probe during the measurement of adipose tissue.

Detection of lameness
Assessment of lameness was based on the observation of the standing cows, with an emphasis on the state of the limb and position of the back at the time of examination.Lamenes was assessed using Zinpro Locomotion Score method used for early detection of hoof disorders, monitoring prevalence of lameness, comparing the incidence and severity of lameness between herds and identifying individual cows for functional hoof trimming.
The method is subjective.353 cows, including the animals in the experiment, were examined prior to the start of the synchronization protocol and insemination in order to have an insight into the presence and expression of lameness in the breeding facility.All obtained data were processed and displayed using the Microsoft Excel 2010 software package.All observed statistically significant differences were evaluated according to "p" values for p <0.05, p <0.01 and p <0.001.The primary processing of results from examined Čengić et al: Success of protocols for synchronization of estrus and ovulation in dairy cows in summer 40 cows was done using descriptive statistics method, and the secondary processing of the results was performed using a two-sample "t" test.The classification of observed statistical significances between the experimental and the control group is marked with stars *, ** and *** depending on the level of the "p" value., 2004).This is confirmed by some other authors (Stevenson et 1996).

Table 1. Relation of cow categories in research
In our case primiparous cows in both groups had a higher conception rate than multiparous cows and it was significantly more pronounced in the control group which is consitent with Sakaguchy's claim.Some studies have shown that the conception rate is best when cows are inseminated within 90-110 days postparum (Waters et al., 2009) which is the period when they are not at peak lactation and have satisfactory BCS.It is often assumed that NEB in early lactation has a negative effect on oocyte quality even up to the first 80-100 days of lactation, thereby reducing the conception rate to the first few inseminations (Patton et al., 2006).Artificial insemination at estrus gives a better conception rate than fixed -time insemination but it also causes more embryonic deaths (Stevenson et al., 1999).
In the period up to 120 days of lactation, the conception rate is up to 40% and it stays at the same level (Thatcher et al., 2000).The results of Thatcher coincide with our results in the case of a control group, where the conception rate after the first insemination after 120 days was 38%.
Cows that have BCS ≥2.75 at the moment of insemination gets pregnant in a higher percentage (Lima et al., 2009), which is observed in the control group that had BCS 3.5 during the first insemination and the success rate of the conception of 38%, indicating the need to establish a BCS assessment protocol before an insemination program.
The conception rate after the first insemination is expected to be in the range of 43-45% for cows with normal puerperium and 15-24% for cows with puerperal disorders (Stevenson et al., 1989).Bearing in mind that monitoring of the health condition found that puerperial disorders were frequent in both groups, a lower conception was understandable, especially after the first insemination in the experimental group.
Holstein  (Stevenson, 2001), but in our case the presynchronization and synchronization protocol (5dCOS2) did not significantly improve the conception rate after the first postpartum insemination.But there was positive effect after the second fixed-time insemination (COS72) as embryonic mortality was decreased in the summer months, when it is usually more pronounced.In the performance analysis, it should be taken into account that COS72 protocol was carried out after more than 4 months postpartum, that it was resynchronization and that the previous protocol was likely to have an effect on P4 elevation that had a positive effect on the dominant follicles, and consequently to a better conception and less embryonic deaths.There are no significant differences in the total number of pregnant cows between the two investigated groups, which indicates the negative impact of all other management factors on the production of all cows regardless of the number and stage of lactation.

CONCLUSIONS
In problematic herds, insemination programs shouldn't start before the end of 90 days lactation period in order to eliminate most of the negative factors which reduce their effectiveness.Presynchronization ahead of synchronization is desirable when introducing a large number of cows into the program for first postpartum insemination.In cows that do not get pregnant after the first synchronized artificial insemination, resynchronization with the selected protocol is sufficient and the use of ovsynch and modified ovsynch protocols results in equalizing the ratio of pregnant primiparous and multiparous cows.
primiparous cows get pregnant after artificial insemination, but they also have a longer service period due to frequent puerperal disorder.Protocol for synchronization protocol and ovulation such as COS72 gives satisfactory results in the summer period for cows with ≥120 days postpartum.Prerequisites for a successful artificial insemination program are good BCS, general health status, reduced lameness, the elimination of heat stress in accordance with the location and farm design, since the negative environmental impacts and management failures considerably lower the success of the first but also of subsequent artificial insemination and extend service period.

Veterinary
Journal of Republic of Srpska (Banja Luka), Vol.XIX, No.1, 35-50, 2019 Čengić et al: Success of protocols for synchronization of estrus and ovulation in dairy cows in summer 38 whether to treat them or to introduce them into the artificial insemination program.The research was conducted from February 2013 to March 2014.The animals involved in the study stayed in the barn, in fenced spaces using tether system.Data about cow age, production, diet, health, reproductive abnormalities and excretions were obtained from the farm protocol or during animal experiments.Hormonal treatment and artificial insemination In order to stimulate ovarian function and accelerate the first ovulation On day15 , postpartum cows in the experimental group received an injection of 100 µg GnRH (Dephérélin Gonavet Veyx®, 1 ml = 0.05 mg or FertagylTM Intervet Schering-Plow Animal Health, 1 ml = 0.1 mg) Protocols for synchronization of estrus and ovulation were divided into presynchronization with two injections of prostaglandin F2α (Estrumate, Schering-Plow Animal Health, 1ml = 250µg Cloprostenol) in 14 days period and synchronization of ovulation 11 days after the last injection of prostaglandins (Presynch 11 + 5dCoS2).The presynchronization protocol was started at 53 ± 1 days postpartum, the synchronization protocol at 67 ± 1 days postpartum, and the first artificial insemination was performed at 77 ± 1 days postpartum.The next day artificial insemination was carried out between 8.00-13.00h.Pregnancy diagnosis was done by ultrasonography (Esaote MYLAB30VETGOLD, LV 513 Veterinary endorectal linear probe 10-4) 42 days after insemination, as earlier embryo can be covered with endometrial folds.Placenta development and the onset of fetal ossification in this period make the diagnosis easier.Non-pregnant cows were subjected to resynchronization with Cosynch 72 protocol, at the end of which they were immediately inseminated.Pregnancy diagnosis was repeated after 42 days.These diagrams present a scheme of hormonal injection and insemination in performed protocols for synchronizing estrus and ovulation.Diagram 1. 5dCoS2 protocol was applied after the process of presynchronization.Diagram 2. COS72 protocol was applied after unsuccessful synchronization with protocol 5dCoS2.Republic of Srpska (Banja Luka), Vol.XIX, No.1, 35-50, 2019 Čengić et al: Success of protocols for synchronization of estrus and ovulation in dairy cows in summer 39

1 . 41 Graph 2 . 42 Graph 4 .protocols for synchronization of estrus and ovulation in dairy cows in summer 43 Graph 6 .
The time interval of days before the first detected estrus, artificial insemination and the length of the service period for all cows within the experimental group, as well as the mean value of the parameters presented with the subtracted and added standard deviation of the mean value (x ± Sx).Days Partus-estrus interval Partus AI interval Servis period Veterinary Journal of Republic of Srpska (Banja Luka), Vol.XIX, No.1, 35-50, 2019 Čengić et al: Success of protocols for synchronization of estrus and ovulation in dairy cows in summer Recorded performance of 1, 2, ≥3.artificial insemination related to all cows from the experimental group, with an average insemination index and the percentage of pregnant and nonpregnant cows until the end of the research.Control Graph 3. The time interval of days before the first detected estrus, artificial insemination and the length of the service period for all cows within the control group, as well as the mean value of the parameters presented with the subtracted and added standard deviation of the mean value (x ± Sx).Republic of Srpska (Banja Luka), Vol.XIX, No.1, 35-50, 2019 Čengić et al: Success of protocols for synchronization of estrus and ovulation in dairy cows in summer Recorded performance of 1, 2, ≥3.artificial insemination related to all cows from the control group, with an average insemination index and the percentage of pregnant and non-pregnant cows until the end of the research.Graph 5.The ratio of the length of the first observed partus-estrus interval and the length of partus -artificial insemination interval between the experimental and the control group.Columns with *** (p <0.001) are statistically extremely significant.The success ratio of the 1st and 2nd artificial insemination with a significant between them.Columns with * (p <0.05) are statistically significant Graph 7. The graph represents interrelations in the appearance of abnormalities of puerperium, the different etiologies between the investigated groups of cows.

Table 2 .
Presentation of the body condition status in the experimental and control group in the period of the first postpartum insemination.

Table 3 .
Percentage of the lame cows on the farm, calculated at the beginning of the research, using the "Zinpro Locomotion Score" method.

Table 4 .
Percentage ratio of pregnancy between the groups at the end of the study period.