1 Introduction

Historically, the geo-ecology of Iran favors small ruminant production and more than 57% of the available animal units of the country are sheep and goats. Moreover, around 2.81% of the total world goat population (921 million) exists in Iran, which reflects the adaptability of this species to the country's hot climate and semi-arid conditions [10, 31] Considering several factors including droughts and shortage of water and food resources, increasing costs of feed, poor risk-return ratio, and rapid urbanization, the rearing system of small ruminants has started to gradually change from the less profitable traditional farming to the industrial and well-managed systems. Moreover, to enhance the efficiency of milk production and the economic profitability of flocks, utilizing high-yielding foreign breeds has been promoted in recent decades [16, 29]. The Murciano-Granadina (M-G) goat is the most important Spanish breed, which was developed in 1975 in semi-arid areas of southeastern Spain by cross-breeding of Murciana and Granadina breeds. The M-G goat breed is well-known for preserving relatively high milk production (up to 500 kg in a 280-day milking period) in hot climates and less favorable regions in terms of nutrient supplementation. This breed has a small to medium-sized body (70–77 cm high at the withers and 30–50 and 50–60 kg body weight in males and females, respectively), covered by uniform black to brown colored skin and hair [24]. The multi-purpose and relatively high milk-producing characteristics of this breed, together with its adaptability to Iran's climatic conditions have resulted in deciding to import this breed to the country since 2015.

Lacking of scientific report about the performance and health complications of this breed, makes investigation of the prevalence, risk factors and therapy of pseudopregnancy (PPG) in the M-G breed does an interesting subject of study, as it’s the most common reproductive disorder of this species [2, 4]. The PPG affects the reproductive and productive efficiency of dairy goats due to increasing the length of the inter-kidding interval through anestrous-caused infertility and also decreased pregnancy rate which results in an extended lactation period. It is defined as the abnormal accumulation of sterile fluid in the uterus, due to failure of luteolysis in the absence of pregnancy [5, 12, 28]. The disorder is also called hydrometra, but technically, PPG is more precise to describe this clinical condition, because hydrometra is also used to describe pathologic accumulation of uterine fluid during pregnancy. The main purpose of this study was evaluating the prevalence of pseudopregnancy in bred M-G does. Moreover, the prevalence of PPG in different age groups, parities, milk production levels and between breeding and non-breeding seasons was compared. The relationships between age, parity, milk production, and prevalence of PP were analyzed. In the second part of the study, the effects of different treatment protocols on the cure rate and subsequent fertility of affected does were evaluated.

2 Materials and methods

This study was done in a large industrial M-G dairy goat farm with a total population of 5000 and 1963 milking goats on average, located in Kerman province, Iran (27° 31′ 30″ N, 57° 52′ 48″ E), from January to August of 2023. The region has a semi-arid, desert-like climate with 409 m’ height above mean sea level and an average annual rainfall of 166 mm. The average days in milk (DIM) of the milking herd was 140 and the daily milk yield per milking goat was 1.5 kg with 5.5% milk fat.

The studied animals were kept in an intensive system composed of several well-equipped closed barns. They were fed a diet based on NRC (2007) recommended ration fulfilling their nutritional requirements which was formulated by using the small ruminant nutrition system software program (SRNS, 2012), using 42:58 as the forage to concentrate ratio. Daily ration of the goats consisted of (as-fed), 0.28 kg alfalfa hay, 1.3 kg corn silage, 0.1 kg wheat straw, 0.1 kg beet pulp, and 0.95 kg of concentrate mixture (34.63% corn, 18.95% barley, 9.55% soybean meal, 9.76% canola meal, 3% soybean whole, 3% meat meal, 11.16% wheat bran, 1.31% fat, 0.58% magnesium oxide, 2.58% sodium bicarbonate, 1.33% calcium carbonate, 2.74% vitamin-mineral premix, 0.88% bentonite, and 0.53% Salt; as fed). Diets were fed as total mixed rations (TMR containing 15.2% of crude protein and 2.46 Mcal of ME/kg of DM) and delivered 4 times daily. Goats had free access to fresh water all the time. They were milked two times a day by a milking machine (GEA® group milking machine, Düsseldorf, Germany) in a standard milking parlor with 96 milking units at 06:00 am and 18:00 pm. M-G does involve in this study ages 1–7 years old (17.25% 1 year old, 39.77% 2 years old, 19.01 three years old and 23.97% ≥ 4 years old) and 20.97% of them were primiparous, 26.69% were secondiparous, 38.29% had parity number of 3 and 14.03% had parity number of four or above, with total average parity number of 2.39. The BCS range of animals was between 3 and 3.5.

According to previous clinical observations, reproductive indices, and hormone analysis profiles, the reproductive season was considered from early August to early February. All does were bred naturally by being transferred to the mating pen and exposed to bucks at mating pen at the ratio of 1:15. Routine reproductive examinations for pregnancy diagnosis were done around 40 days after termination of being exposed to bucks, through abdominal ultrasonography (US) of standing positioned animals by using of a B-mode ultrasound device (iScan®, DRAMIŃSKI CO, S.A., Gietrzwałd, Poland) and a curved probe at the frequency of 2 MHZ. During the time of the study, bred does (N = 1310) were tested by US, and provided results were recorded precisely. The diagnosis of PPG was based on lacking a view of any fetal or placental tissues, together with the presence of anechoic areas separated by double layered sack-like structures created by opposing walls of the distended, curved uterine horns in ultrasonography. The clinical signs of PPG, including abnormal bilateral abdominal distension due to the accumulation of a variable amount of intrauterine fluids, were also considered for diagnosis. Diagnosed cases were reexamined two weeks later to confirm the occurrence of the disease.

In the second part of the study, two experimental groups (n = 12) of examined goats from those diagnosed as pseudopregnant were randomly selected. Group one was treated with two dosages (100 µg per animal, i.m.) of cloprostenol sodium (Clo PG®, Rooyan darou CO, Semnan, Iran) 11 days apart [27], while group two received the same treatment together with antibiotic therapy by intramuscular administration of procaine penicillin (20000 IU/kg) and dihydrostreptomycin (20 mg/kg, Penstrep-400®, Interchemie CO, Netherland) for three consecutive days from the time of diagnosis according to the recommended dosage [27]. To assess cure rates in both groups, re-examination by US was done 36 h after the second prostaglandin injection and cured cases were rebred and rechecked to show their pregnancy status after rebreeding.

Analysis of data was done by chi-square test and SAS 9.2 software (SAS Inst., Inc., Cary, NC, USA). Effects of age and milk yield on the prevalence of PPG were analyzed by logistic regression through proc logistic Moreover, the prevalence of PPG between different age groups, parities, daily milk production levels, and during and out of breeding season was compared by Chi-square test. Average daily milk production level was compared between different parties, and between normal and pseudopregnants by ANOVA through PROC GLM.

3 Results

The results of ultrasonography showed that 118 out of 1311 cases (9%) were pseudopregnant. An ultrasonography of a pseudopregnant ewe is shown in Fig. 1. The occurrence of PPG was accompanied by abnormal abdominal enlargement in most cases and the resolving of this clinical sign occurred soon after cloudburst, following treatment by prostaglandin (Fig. 2). The average age of diseased goats was 3.4 ± 1.35, while it was 3.28 ± 1.55 for non-affected does (P = 0.05). The prevalence of PPG varied between different age groups (P = 0.0003) and parities (P < 0.0001), Table 1. The lowest (3.1%) and highest (14.46%) prevalence of PPG occurred in 1 and 3 years old does, respectively. Moreover, differences between various age groups were significant except for 2 and ≥ 4 years old does. Considering different parities, the highest prevalence of PPG occurred in parity ≥ 4 (16.8%) followed by secondiparous does (14%), while it was significantly lower among primiparous (3.63%) and those with parity number of 3 (5.57%). Regardless of these differences, based on the results of logistic regression, no significant relationship between increasing age and prevalence of PPG was found, in general (P = 0.72, OR 0.97, CI 0.86–1.1).

Fig. 1
figure 1

Ultrasonographic view of uterus in a case diagnosed as pesudoprenant. Accumulation of fluids (hydrometra) together with lacking of any placental and fetal tissues

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Fig. 2
figure 2

Abnormal abdominal enlargement in a pseudopregnant doe (Left) before treatment by prostaglandin and resolving this condition after cloudburst (Right)

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Table 1 Incidence rate of pseudopregnancy in different age groups, parities and levels of milk production
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According to logistic regression provided results, the relationship between increasing daily milk yield and prevalence of PPG was not significant generally (P = 0.2, OR 1.41, CI 0.93–1.4), but a significantly different prevalence of PPG was found between different milk production groups (P < 0.0001). The highest (15.82%) and lowest (5.17%) prevalence of PPG occurred in 1–2 kg and less than 1 kg daily milk yield group, respectively. Moreover, the average level of milk production itself, varied between different age groups (P < 0.0001), and 1-year old does (1.671 ± 0.061) had higher milk production compared to other age groups. Comparing milk production between Non-PPG (1.4235 ± 0.027, 95% CI [1.3923, 1.6812]) and diseased does (1.5368 ± 0.07, 95% CI [1.3702–1.4767]) showed that the difference is tended to be significant (P = 0.07). Differences in daily milk yield between PPG and Non-PPG were not significant in 1–2 and ≥ 4 years old does (P = 0.7), but it was significant in 3–4 years old does (P = 0.04), Tables 2 and 3.

Table 2 Incidence rate of pseudopregnancy in different production level in each group
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Table 3 Average daily Milk yield of different age group of does, according to the incidence of pseudopregnancy
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Analysis of the prevalence of PPG in each production group in different age groups and also in each age group with various production levels separately, showed that the difference in the rate of PPG was not significant in age groups 1–2 and 2–3, while it was significantly increased by elevating milk production in higher age groups, moreover the rate of PPG significantly increased by age only in higher production group, since different ages with production level of ≤ 1 kg/day showed no significant difference. The prevalence of PPG was quite similar in (8.9%, 80:891) and out (9%, 38:420) of the breeding season. In the second part of the study, the cure rate in the first group was 66.8% (8/12), while it was 75% (9/12) in the second group (P = 0.6). Moreover, 75% (6/8) and 88.8% (8/9) of recovered cases in the first and second groups were pregnant after rebreeding, respectively (P = 0.45).

4 Discussion

Pseudopregnancy (PPG) is an important pathological condition, as it represents one of the main causes of temporary infertility in dairy goats [23, 26]. The common duration of PPG is not constant, and ranged from 70 to 180 days, but it is usually longer than normal gestation period [9, 18]. Five grades for PPG were defined according to intensity of uterine fluid accumulation from 0 (Normal uterus in absence of fluid) to 1 (presence of a small volume of anechoic fluid), 2 (presence of fluid-filled sac), 3 (presence of a large volume of fluid) and 4 (presence of a large amount of fluid which occupies whole image of ultrasonography) [19]. As PPG may occur irrespective of history of mating or pregnancy, the exact pathophysiology of this disease remains to be known clearly, but the pivotal factor for developing PPG is the persistency of corpus luteum which results in high plasma progesterone level and anestrous [28]. Failure of luteolysis may occur as a result of hormonal disturbances caused by exogenous (Induction of estrus and ovulation by GnRH analogs or consuming phytoestrogens) or endogenous (ovarian cysts, high level of prolactin) sources, or lack of PGF2ɑ secretion due to embryonic mortality after maternal recognition of pregnancy, around day 40 of pregnancy [6, 17, 18, 30, 33]. In this study, we investigated the prevalence rate of PPG and its main risk factors in Murciano-Granadina dairy goats in Iran. Moreover, in the second part of the study, we investigated the effects of different treatment protocols on the subsequent fertility of affected does.

As far as we know, no previous study reported the prevalence rate of PPG in the M-G breed does, so far. Moreover, we also didn’t find any report investigating the prevalence rate and risk factors of PPG in dairy goats in Iran, at least in this population scale. The reported range for the prevalence of PPG is from 1.37% to 30.4%, but most previous studies reported a rate of 9% to 12% which is in accordance with the prevalence of PPG in our study [1, 3, 4, 7, 18, 21]. Several factors including breed and genetic predisposition, age, milk production, pregnancy loss, reproductive management, days in milk, systems of production, and nutritional management were reported to be associated with PPG occurrence [1, 13, 15, 21, 32].

Three is a consensus about higher prevalence of PPG in aged does, but inconsistent finding about its relationship with parity exist [1, 13, 21, 32, 33]. The persistence of corpus luteum is prevalent after the second year of lactation without being mated [23]. In this study, the average age was higher in the diseased does compared to the normal group and significant differences in the rate of PPG existed between various age and also parity groups. In fact, similar to most previous reports, the lowest rate of PPG was found in the age group of 1–2 and primiparous does and the prevalence rate was elevated by increasing age and parity, except for parity number of three and age of ≥ 4. Moreover, no significant relationship between age (and parity) and prevalence of PPG was found generally, which can be explained by a surprisingly lower prevalence of PPG in does with a parity number of three and an age of ≥ 4. This can be explained by changing prevalence in certain ages and/or milk production groups, and lacking of a significant increasing trend by elevated age and milk production. Moreover, considering the recurrent nature of the disease and its long-term negative impacts on reproductive efficiency [22], the policy of the herd for culling aged does with reproductive problems and a history of infertility may affect the prevalence of PPG and result in lower prevalence in aged does with high parity number, and cause this discrepancy. Controversial reports about the association between milk production and PPG were presented [1, 14, 33]. Lacking a significant relationship between milk production and prevalence of PPG despite tending to be significant or significantly higher milk production), among 2–3 years old does (P = 0.08), 3–4 years old (P = 0.04) PPG does, and in general (P = 0.07) indicates that relationship between milk production and PPG is significant only in certain ages. The lowest and highest prevalence of PPG occurred in does with less than 1 and 1–2 kg of milk production per day, respectively and this can be explained by the effect of age, as the high production are younger and have a lower risk of developing PPG. Significantly increased prevalence of PPG by elevating milk production in higher aged groups compared to young does and also in aged does only with production level of higher than 1 kg/day, indicated higher sensitivity of aged does to develop PPG by increasing level of production and the effect of milk production level on increasing rate of PPG, in ages does. This finding may be related to metabolic complications of aged does with high milk production, which is suggested to be investigated in future research.

There are some reports about a higher prevalence of PPG in cases of cyclicity out of the breeding season [3, 8]. Moreover, a positive genetic correlation between out-of-season kidding ability (and also extended lactation and whole-life days in milk, as a result) with PPG was reported. The persistence of milk production in M-G goats is heritable [25], and based on the explained findings, this may affect the genetic predisposition of individuals to PPG in this species. According to our provided results for M-G does, there was no difference in the prevalence of PPG during the breeding season and out of it, which is in agreement with recently published reports [1]. This might have occurred due to the low geographic latitude of the place where the studied farm is located, which diminishes does' seasonality.

Pregnancy rate following treatment of pseudopregnancy is usually lower than normal level. Besides the negative impacts of endocrine disturbances due to failure of luteolysis, inadequate uterine involution and partial resolving of intrauterine fluid accumulation and endometritis may also play role. Moreover, the high level of progesterone of inhibits uterine immune response in ruminants [11]. A standard therapy regimen of repeated prostaglandin administration 11–12 days apart, increased the rate of successful treatment and improved post-therapy pregnancy rate by reducing the chances of disease reoccurrence. It's supposed that drainage of the remnant uterine fluids by second injection, following induction of luteolysis and cloudburst by first injection is responsible for this improved response to therapy. The PPG itself is defined as a sterile fluid accumulation in the uterus, but as it imitates pregnancy, resolving it through cloudburst mimics parturition and may predispose sterile uterine environment to bacterial contamination, which can be detrimental due to suppressed uterine immunity as a result of chronic high plasma progestogen concentration. Considering this hypothesis, adding prophylactic antibiotic therapy to this treatment regimen may improve pregnancy rate after treatment. The cure rate in the first and the second groups of the study was 66.8% (8/12) and 75% (9/12) respectively. After rebreeding, the pregnancy rate was 75% (6/8) and 88.8% (8/9) in the recovered cases of the first and second groups of the study. The cure rate of 59% and pregnancy rate of 48% following two prostaglandin injections were reported before [14], which is lower than our finding. In another study, despite a 95% cure rate after the second prostaglandin administration, a pregnancy rate of 45% following treatment with three doses of prostaglandin was reported [20], another study reported conception rate of 64%, which is comparable to our result [3]. Despite numerical improvement in the cure rate and post therapy pregnancy rate compared to standard treatment group, systemic antibiotic administration didn’t have any significant impact, and this might be due lacking of considerable intrauterine infection or small number of observations in each group of the study.

5 Conclusion

The prevalence rate of pseudopregnancy in Murciano-Granadina dairy goat in this study was 9%, which is following other dairy goat breeds. Higher than average daily milk yield, is contributing to increased prevalence of this disease, especially in higher ages. The effect of the breeding season was not significant. Moreover, despite lacking significance due to the small size of the study, adding an antibiotic to the treatment regimen of administrating two doses of prostaglandin 11 days apart, improved the cure rate numerically by 7.8% and the post-treatment pregnancy rate by 13.8%, but due to failure of inducing significant impacts, it’s not recommended yet to use prophylactic antibiotic therapy for prevention of uterine infection in the treatment regimen of PPG, and it needs further studies.