2.1. Description of the Study Area

The study was carried out at Ameya district South West Shoa Zone, Oromia region. The district is located 144 km southwest of Addis Ababa with altitude ranging from 1850 to 2800 m above sea level. The area is part of the central highlands of Ethiopia. The average annual minimum and maximum temperatures were 15° and 24°C, respectively. The annual rainfall ranged from 1350 to 1600 mm. The area receives bimodal rainfall with two rainy seasons in a year.

2.2. Experimental Animals and Their Management

All animal handling and experimental procedures were approved by the Animal Care and Use Committee of the Jimma University College of Agriculture and Veterinary Medicine.

The study animals were selected from different dairy farms. Dairy herds were selected based on their uniformity in the management system and the willingness of the farmer to participate. Accordingly, a total of 120 dairy cattle, 60 Horro (30 cows and 30 heifers) and 60 Holstein-Horro cross-breed (30 cows and 30 heifers), were enrolled in the study. All cows were greater than 60 days postpartum and in parities 1–4. The study animals were selected based on selection criteria such as overall health, presence of mature corpus luteum, absence of pregnancy, and body condition score (BCS). The BCS ranged from 2 to 4 on a scale of 1 to 5. Rating the BCS was done subjectively based on the fat cover and flesh over the ribs, loin, and tail head [30]; BCSs were evaluated through optimal when ≥ 3 BCS and suboptimal when < 3 BCS [31]. The reproduction status of cows and heifers was confirmed based on the information from the owners and through internal reproductive organ palpation. The selected animal’s reproductive organs were palpated per rectum to assess pregnancy and the presence of corpus luteum on either of the ovaries.

2.3. Study Design

Candidate animals were divided into two by breed (Horro and Holstein-Horro cross-breed) and within breed by parity (cows and heifers). During the commencement, all animals that have a CL on either of their ovaries were injected 2 mL of PGF2α (Synchromate, Bremer Pharma GMBH, Germany, 1 mL solution of Synchromate containing cloprostenol 0.263 mg equal to cloprostenol 0.250 mg) intramuscular. The animals that received the hormone were closely observed for any sign of estrus for five consecutive days. Any signs of estrus (restlessness, redness of vulva, mounting others, standing to be mounted, vaginal mucus discharge, frequent urination, sniffing, bellowing, and other related signs) were recorded based on visual observation and information from the owners. Animals that exhibited estrus were randomly allocated to be inseminated by SS or CS. Insemination was done 12–18 h after the onset of estrus. The number of pregnant cows and heifers following AI was determined by the number of animals that exhibited estrus and were subsequently inseminated. Pregnancy diagnosis was conducted by rectal palpation 60 days after AI.

2.4. Statistical Analysis

Statistical analyses were performed with SAS (Version 9.4, SAS Institute Inc., Cary, NC). Estrus expression and conception rate data were analyzed using binary logistic regression while data on the interval to estrus were analyzed using a general linear model, and the significance level was set at p < 0.05.

3.1. Estrus Response to PGF2α Hormone

The results of the estrus response rate to PGF2α hormone in the study area are presented in Table 1. When PGF2α is administered to animals with a functionally mature corpus luteum, 85% to 95% are expected to reach estrus within 7 days of treatment, and 70% to 90% show estrus signs 3 to 5 days after treatment [32]. In this study, 80% (96/120) of animals responded to the PGF2α hormone treatment. This consisted of 19 Horro cows and 25 heifers and 24 Holstein-Horro crossed cows and 28 heifers. The logistic regression analysis indicates parity’s significant effects on the estrus response rate. As a result, heifers were 2.99 times more likely to respond to the treatment than cows (OR = 2.99; 95% CI: 1.13–7.87). This indicates that the efficacy of the treatment regime is better in heifers than in cows in the study area. This might be associated with differences in their reproductive physiology, differences in body condition, and the presence of functional corpus luteum at the time of injection. Additionally, it could be due to the management system in general and the feeding system in particular. In the highlands of Ethiopia, smallholder dairy farmers feed their animals mainly natural pasture, crop residues, and nonconventional feeds [33, 34] which are generally poor in nutritional value. As a result, if no compensatory intake of nutrients is achieved to cope with the requirement for multiparous cows, reproductive functions, including hormone synthesis and secretion, as well as follicle ovulation may be depressed and thereby reduces the response in the overall reproductive performance [35, 36]. Higher estrus responses in the nulliparous females in this study corroborate these earlier findings of Chanyalew et al. [37], and Tadesse et al. [38] obtained higher estrus response in heifers as compared to cows in similar management systems; also, Bonato et al. [36] obtained a lower rate of estrus in primiparous (61.36%) compared to nulliparous (76.39%) females in Nelore cattle. In contrast, Haile et al. [24] reported a higher proportion of estrus response in multiparous cows as compared to heifers using a similar approach in a similar management system. Diaz et al. [39] also obtained similar results in Brown Swiss cattle. The differences could be attributed to various factors such as the management systems used by dairy farmers before the estrus synchronization protocol, the genetic factors of the animals, and the type and efficacy of drugs used compared to other studies.

Even though there was no breed effect on estrus response (p > 0.05), numerically higher proportion (86.67%) of estrus response was recorded in crossbreds compared to local breeds (73.33%) in those who received a similar dose of hormone injection. This may be attributed to variations in hypothalamic, pituitary, and ovarian functions between Zebu and Bos taurus cattle, resulting in differing fertility levels between the two species, even when subjected to the same feeding conditions [40, 41]. Similarly, Haile et al. [24] observed a higher estrus response rate (100%) in cross-breed animals than local (86.7%). Gugssa et al. [42] and Tadesse et al. [38] also reported similar results. In the current study, the BCS significantly influenced (p = 0.001) the estrus response rate among the animals that received the hormone injection. Those suboptimal BCSs were 0.17 times less likely to respond to the hormone than their counterparts (OR = 0.17; 95% CI: 0.06–0.47). This is in line with the fact that greater BCS and positive energy balance caused greater estrus expression [43]. BCS is associated with ovary functions, and animals with higher BCS produce larger dominant follicle size and improve ovulation rate, and thereby estrus response rate [44, 45]. Quintana-Utra et al. (2019) found that a significant percentage of cows in the group with a BCS lower than 2.5 exhibited conditions compatible with anestrus, indicating a state of reproductive inactivity characterized by a lack of cyclical activity and reduced ovarian function. They associated it with nutritional deficiencies (proteins, vitamins, minerals, and lipids) due to its relation with body condition. This reflects the situation in Ethiopia, particularly the smallholder farming system, where undernutrition or inadequate intake of nutrients relative to metabolic demands is a major factor hindering livestock production systems, particularly among animals dependent upon natural forages for most or all of their feed requirements [47, 48]. The result of the current study is in agreement with the report of Kebede et al. [49] who also obtained greater estrus response in cows and heifers with optimal BCS than those with suboptimal BCS. Richardson et al. [50] also noted a decreased expression of estrus in under-conditioned animals compared to animals in moderate conditions.

3.2. Time Interval to Estrus After PGF2α Injection

Regardless of whether cows are inseminated during natural or synchronized estrus, the timing of AI is determined by the onset of estrus, based on the assumption that the interval between the start of estrus and ovulation remains consistent across animals. Inaccurate timing of insemination in relation to estrus onset and ovulation is one of the key factors that limit optimal conception rates in cattle breeding, especially in smallholder farming systems where record-keeping is often lacking. Hence, once a response to PGF2α is observed, understanding the timing of estrus onset becomes crucial, as the interval between the onset of estrus and ovulation can significantly influence the success of AI [51]. The results of the interval to estrus after PGF2α injection in the study area are presented in Table 2. In this study, the interval from PGF2α injection to the onset of estrus was significantly affected by breed (p < 0.05), while the effect of parity and BCS was not significant. Regarding the breed, a longer time interval was observed in Horro than in Holstein-Horro crossed animals. The interval to estrus after PGF2α depends on the stage of the estrous cycle at the time PGF2α is administered [52]. PGF2α is only effective in diestrus animals meaning it is only effective in the presence of a functional CL from Day 7 to Day 17 of a normal estrous cycle (estrus = Day 0) [53]. As a result, PGF2α administration in midcycle (Day 8 to Day 11) or later in the luteal phase (Day 12 to Day 15) results in a mean time to estrus of 70 and 62 h, respectively [32]. In general, while luteolysis can be effectively synchronized by administering PGF2α during the diestrus period, the onset of behavioral estrus may occur anywhere from 2 to 5 days later. This variability is attributed to different factors such as the estrous cycle stage at the time of PGF2α treatment [32], differences in the size of the dominant ovarian follicle at the time of injection [54], the time required for an ovulatory follicle to develop [55], dosage of PGF2α injected [56], differences among cows in the rate of regression of the CL following treatment [52], and nutrition and environment [57]. Breed is another factor influencing the time interval from PGF2α injection to the onset of heat [58, 59]. The delayed onset of estrus following PGF2α in local breeds as compared to their counterparts could be associated with hormonal factors such as differential response to PGF2α between B. taurus and B. indicus [60], reduced sensitivity to PGF2 α in luteal tissue in B. indicus cattle compared with B. taurus [59, 61], and amount of released estrogen and time response to estrogen hormone [62]. This study produced results that corroborate the findings of a great deal of the previous work in this field. Gugssa et al. [42] found that 50% of the animals exhibited estrus within 72–96 h, while 40% within 48–72 h, regardless of breed and parity. Demis et al. [63] and Ejigayehu and Alemayehu [64] also reported a similar range of 77.82 ± 2.74 h and 74.53 ± 5.16 h, respectively, in identical management systems. The reports of Dodicho et al. [65] were also in close range (62.4 ± 4.59 h) for cows using a single shot of PGF2α. In the same study, however, heifers responded in shorter intervals (54.35 ± 4.34 h) than heifers in this study (71.75 ± 2.3 h). Chanyalew et al. [37] also recorded that most of the animals come to estrus for more than 96 h.

3.3. Pregnancy Rate to Sexed and CS

The results of the pregnancy rate of PGF2α hormone–treated animals in the study area are presented in Table 3. In this study, out of 96 Horro and Holstein-Horro cross-breed cows and heifers that responded to PGF2α, 58 animals (60.4%) became pregnant. Out of these pregnant animals, 25 of them were Horro cows and heifers and the remaining 33 were Holstein-Horro crossed cows and heifers. Thus, the overall pregnancy rate was 60.4%. Except for semen type, the others including breed, parity, and BCS did not influence the pregnancy rate in the current study. The pregnancy rate was significantly higher in using CS (35/48 = 72.9%) than in SS (23/48 = 47.9%). In general, P/AI in animals inseminated with CS has been found greater than that of animals inseminated with SS. This could be due to fewer numbers of sorted sperm per straw [66], the process of sperm sorting often leads to some damage to sperm structure [67], and reduced lifespan of SS in the female reproductive tract [13]. The present findings seem to be consistent with other research which found P/AI using SS at first service 47% for Holstein heifers and 53% for Jersey heifers [68], overall pregnancy rate of 51% for Holstein-Friesian heifers in commercial dairy herds [69], 41% to 53% across semen type and dosage on Holstein heifers [70], 46.8% pregnancy rates in Holstein dairy cows [71], and 46.1% pregnancy rates for Boran and Boran X Holstein cross cows and heifers [72]. The fertility of SS in this trial was significantly greater than that observed in the study by Karakaya et al. [15], who observed a conception rate of 31.8% to 25.7%, and Healy et al. [20], who reported 31.6% conception rates in nulliparous Holstein heifers, 39% mean conception rate for heifers [73], and 21.0% average pregnancy rate for lactating Holstein-Friesian cows [21] using SS. In contrast, higher fertility using SS was also recorded in this study. Correa-Calderón et al. [23] reported a 65% conception rate in winter at Day 35 post-AI in Holstein heifers, 53.9% to 66.3% pregnancy rate using deep intracorneal insemination [74], and 80% (388/485) pregnancy rate in the dairy village production system in Central Ethiopia [75]. The differences observed in the current and previous works could be due to selection criteria used to select the most fertile cows, dam parity and season when AI was performed, semen dosage, insemination techniques used, and overall management system.