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Full Research Article

Effect of Treatment with Equine or Human Chorionic Gonadotropin on Blood Count Values in Early Gestation in Nulliparous Sheep in Northern Mexico

J
Jesús Armijo-Nájera1
A
Ariadna Vanessa Alvarado-Espino2
F
Francisco Gerardo Véliz-Deras3,*
https://orcid.org/0000-0002-5105-1508
I
Irene Concepción Chavarría-Neri3
G
Gerardo Arellano-Rodriguez3
J
Jessica Maria Flores-Salas3
O
Oscar Ángel-García3
C
Cayetano Navarrete-Molina3
F
Francisco Gerardo Véliz-Romero1
A
Alan Sebastian Alvarado-Espino3,*
https://orcid.org/0000-0003-4590-1673
1Graduate Program-Agricultural and Livestock Sciences, Antonio Narro Agrarian Autonomous University, Laguna Unit, Torreon Coahuila 27054, Mexico.
2Faculty of Veterinary Medicine and Animal Science Campus 2, Autonomous University of Chiapas, Tuxtla Gutiérrez, Chiapas 29050, Mexico.
3Regional Division of Animal Science, Antonio Narro Agrarian Autonomous University, Laguna Unit, Torreon, Coahuila 27054, Mexico.

ABSTRACT

Background: Hormonal treatments can influence hematological profiles through direct and indirect mechanisms in sheep’s. The aim was to evaluate the effect of administering equine or human chorionic gonadotropin (eCG, hCG, respectively) at 7 or 14 days post-mating on blood count values in early pregnancy of nulliparous ewes.

Methods: A total of 63 ewes were used, which were randomly assigned to five experimental groups (EG): EG-eCG7 (300 IU of eCG, 7 days post-mating (DPM)), EG-hCG7 (300 IU of hCG 7 DPM), EG-eCG14 (300 IU of eCG 14 DPM), EG-hCG14 (300 IU of hCG 14 DPM) and; EG-Control (without hormonal treatment). Hematologic variables were determined on days 7, 14 and 28 DPM and included white blood cell count (WBCC), red blood cell count (RBCC), hematocrits (Ht), hemoglobin concentration (HbC), mean corpuscular hemoglobin (MCHb), mean corpuscular hemoglobin concentration (MCHbC) and medium corpuscular volume (MCV). An ANOVA and Pearson correlation were performed on the response variables.

Result: The results showed differences in WBCC on day 7 between the EGs and between days for the EG-Control. The RBCC variable only showed differences between the days of the EG-eCG14. The Ht variable reported differences between the days of the EG-eCG14. Regarding HbC, differences were determined between the EGs for days 14 and 28, as well as between the days of the EG-hCG7. Additionally, positive and negative correlations were evidenced between the variables considered. Based on the above, it is concluded that in the first month of gestation, the variables WBCC, RBCC, Ht, HbC and MCV increase in pregnant ewes. The aforementioned contributes to determining ewes’ reproductive performance and improving their productive and reproductive parameters.

INTRODUCTION

One of the main factors affecting the productivity and profitability of sheep herds is reproductive efficiency (Magaña-Monforte et al., 2024). In this context, hormonal synchronization protocols are commonly used to improve reproductive efficiency (Ashour et al., 2018; Murphy, 2012; Sanchez-Ramos et al., 2022). These protocols include the application of equine chorionic gonadotropin (eCG) or human chorionic gonadotropin (hCG) to stimulate follicular development and ovulation (Ashour et al., 2018; Husein and Haddad, 2006; Murphy, 2012). However, hormonal treatments can influence hematological profiles through direct and indirect mechanisms (Bamerny et al., 2022; El-Sayed et al., 2025). This is especially important, as hemograms provide an overview of the physiological and nutritional status of sheep across reproductive stages and facilitate the diagnosis of conditions (Bamerny et al., 2022; Bhat et al., 2017; Mauffré et al., 2016; Porcu et al., 2020; Rabea et al., 2024). In this sense, hematological parameters such as white blood cell count  (WBCC) “leukocytes”, red blood cell count (RBCC) “erythrocytes”, hematocrits (Ht), hemoglobin concentration (HbC), mean corpuscular hemoglobin (MCHb), mean corpuscular hemoglobin concentration (MCHbC) and medium corpuscular volume (MCV) undergo dynamic alterations throughout pregnancy (Rabea et al., 2024). These fluctuations are usually attributed to hemodilution, fetal metabolic demands and changes in plasma volume (Bamerny et al., 2022; Rabea et al., 2024).
       
Regarding the use of these hormones in hormonal treatments, eCG has a long biological half-life due to glycosylation (Murphy, 2012). And that its application increases twin birth rates, by promoting multiple ovulations through its dual activity, similar to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (Martinez-Ros and Gonzalez-Bulnes, 2019; Porcu et al., 2020). Although eCG is used for reproductive purposes, its interaction with homeostatic mechanisms justifies the monitoring of the number of leukocytes (Bizjak et al., 2018; Gallagher, 2013). This is important, as fluctuations in MCV, MCHb and MCHbC indicate physiological changes during hormonal protocols (Porcu et al., 2020). In this regard, eCG synchronization doses, ranging from 350 IU to 500 IU, have been reported to induce estrus without altering systemic health (Husein and Haddad, 2006; Sanchez-Ramos et al., 2022). In other research, including studies on glycogenic and eCG formulations, results showed that while high doses of other substances affect HbC and MCV, hormonal treatments typically keep rates within physiological limits (Husein and Haddad, 2006; Porcu et al., 2020; Sanchez-Ramos et al., 2022). This suggests that the use of hormonal reproductive protocols with eCG are physiologically compatible with the health of sheep (Dimauro et al., 2008; Martinez-Ros and Gonzalez-Bulnes, 2019; Porcu et al., 2020; Wildeus, 2000).
       
Considering the use of hCG, in the hormonal protocols of small ruminants, it has been reported that it works as an analogue of LH (Adeyeye and Ate, 2016; Aiche et al., 2020; Khan et al., 2009) and that its main application consists of the synchronization of estrus and the induction of ovulation in sheep and goats (Quintero et al., 2015). In this respect, the application of hCG stimulates the formation of accessory corpus luteus, which improves the endogenous secretion of progesterone (P4) (Adeyeye and Ate, 2016; Khan et al., 2009; Lashari and Tasawar, 2010). Additionally, it has been reported that hCG administration, on day 12 post-mating, aims to increase P4 concentrations to prevent luteolysis (Quintero et al., 2015). This facilitates embryo growth and favors placentation (Cam and Kuran, 2004), correlating with higher pregnancy and fertility rates in various sheep breeds (Quintero et al., 2015). In addition, research on the impact of hCG on the hemogram provides data for the clinical follow-up of sheep under intensive reproductive management (El-Sherif and Assad, 2001; El-Sayed et al., 2025).
       
Therefore, the correlation between the administration of eCG or hCG and immediate hematological changes in sheep requires additional research to improve animal health monitoring standards (Mattos et al., 2011; Murphy, 2012). These assessments can clarify how these hormones influence systemic variables beyond the hypothalamic-adenopituitary-ovarian axis (Sanchez-Ramos et al., 2022; Wei et al., 2016). In small ruminants (i.e., sheep and goats), metabolic adjustments associated with hormonal mani-pulation and physiological stress can lead to variations in leukocyte and neutrophil counts (Cismaru et al., 2024; Darwish and El-Ebissy, 2019). Therefore, integrating hematological monitoring into routine reproductive management improves the well-being and productivity of sheep herds (Porcu et al., 2020; Wei et al., 2016). Under-standing the relationships between hormone application and hematological monitoring is necessary to refine protocols used in global sheep production (Gãvan et al., 2010; Greguła‐Kania et al., 2020). Therefore, the objective of this study was to evaluate the effect of hCG or eCG administration at 7 or 14 days post-mating on blood count values in early gestation in nulliparous ewes. In order to contribute to improving the reproductive success of sheep, which will improve the income of sheep farming families, while promoting a sustainable vision aligned with the fulfillment of the sustainable development goals of the 2030 Agenda (Autukaitė et al., 2021; Navarrete-Molina et al., 2024; Pampori et al., 2025).

MATERIALS AND METHODS

General
 
All experimental procedures and animal management used in this study were performed in accordance with international and national standards of ethics, care and animal welfare for research (FASS, 2010; NAM, 2002). The period during which the experiment was conducted included the days between September 15 and December 4, 2025. Additionally, this investigation had institutional approval, with reference number UAAAN-UL-18-3060.
 
Location and environmental conditions of the study area
 
The sheep production unit, where the study was carried out, is an intensive farm in the Comarca Lagunera (25°37′
N, 103°16′  W, 1113 meters above sea level) in north-central Mexico. The region is classified as a semi-arid ecosystem, registering an average annual temperature of 23.8oC, with maximums of 41.0oC in summer and minimums of -1.0oC in winter and an average annual rainfall of 230 mm. Additionally, it registers relative humidity ranging from 12% to 61% and photoperiods ranging from 13 h 41 min during the spring solstice (June) to 10 h 19 min during the winter solstice (December) (CONAGUA, 2025; INEGI, 2010).
 
Animals and experimental design
 
In this study, 63 Dorper × Blackbelly and Dorper ×  Katahdin crossbred nulliparous ewes, 7 to 9 months old, with a live weight of 32.2±4.4 kg, were used. At the beginning of the experiment and to synchronize the onset of estrus and ovulation, all ewes were administered two doses of prostaglandin F‚ α (75 μg of D-cloprostenol sodium; Estrumate®, Darmstadt, Germany) with an interval of 12 days between applications. After the manifestation of estrus, the ewes were exposed to sexually active Dorper males for seven days to ensure mating. For this, the males were marked with vegetable paint in the sternal region to easily identify the mounted females. The day on which the painted sheep of the lumbar region was observed for the first time was considered the day of mating.
       
Based on the above, the ewes were randomly assigned to five experimental groups (EG): EG-eCG7 (n = 14), the ewes in this group received 300 IU of eCG (Folligon®, Darmstadt, Germany) intramuscularly (IM) 7 days post-mating; EG-hCG7 (n = 13) females included in this group received 300 IU via hCG IM (Chorulon®, Darmstadt, Germany) 7 days post-mating; EG-eCG14 (n = 13) received 300 IU of eCG via IM 14 days post-mating; EG-hCG14 (n = 13) received 300 IU via IM of hCG 14 days post-mating and; EG-Control (n = 10) who did not receive post-mating hormonal treatment. In addition, on days 7 and 14 post-mating, all ewes that did not receive hormonal treatment received 1.5 mL of sterile injectable solution (Isosol MDKR, Mediker, Gomez Palacio, Durango, Mexico). The sheep were fed a diet based on food residues from a highly industrialized dairy cattle production system twice a day (1000 and 1800 h). Likewise, all females had ad libitum access to drinking water, mineral salts and shade. To guarantee the physiological status of the females (i.e. pregnant), a diagnosis of pregnancy was made at 32 days post-mating by transrectal ultrasono-graphy, which was confirmed by visualization of intrauterine fluid (embryonic vesicle) and the presence of echogenic structures within anechoic cavities compatible with viable embryos, for which an ultrasound (Chison ECO5 Doppler®, Wuxi, Jiangsu, China) and complemented with a rectal transducer (7.5 MHz, Color® Doppler, Wuxi, Jiangsu, China) was used, confirming that 100% of the experimental units that were included in the EGs were pregnant.
 
Blood sampling and quantification of the values of the blood count to be evaluated
 
On days 7, 14 and 28 post-mating, blood samples were taken from all ewes (n = 63) to determine blood count values. To this end, at 08:00 h on an empty stomach, samples of 5 mL of blood were obtained by jugular venipuncture with BD Vacutainer® tubes of 9 mL (Franklin Lakes, New Jersey, USA). The tubes contained 7.2 mg of dehydrated K2EDTA in their inner walls. This compound acts as an anticoagulant by binding calcium ions and interrupting the blood-clotting process. To continue the analysis of the samples, the hemogram variables considered in the study were quantified. For this, an automated hematological analyzer Hemalyzer HVET 2000 brand HLab (Denver, Colorado, USA), was used. All procedures were performed by a trained technician. The blood response variables considered in the study included: white blood cell count "leukocytes" (WBCC, ×10^9 cells L-1); red blood cell count "erythrocytes" (RBCC, ×10^12 cells L-1); hematocrits (Ht, %); hemoglobin concentration (HbC, g dL-1); mean corpuscular hemoglobin (MCHb, pg); mean corpuscular hemoglobin concentration (MCHbC, g dL-1) and medium corpuscular volume (MCV, fL). All analyses were performed in the Bromatology Laboratory of the Antonio Narro Agrarian Autonomous University, Laguna Unit.
 
Statistical analysis
 
The Kolmogorov-Smirnov test at a confidence level of 95% was used to verify the normality of the data. Considering that the response variables RBCC, Ht, MCHbC and MCV did not show a normal distribution, a Box-Cox transformation was performed. Equality of variances was assessed using Levene’s test with a 95 % confidence level. Subsequently, an analysis of variance was performed, using Fisher’s LDS method to compare means, considering a 95% confidence level. Additionally, the mean and standard error were calculated for each response variable. Finally, Pearson’s correlation coefficient was computed between the independent variables (i.e., experimental group and day) and the dependent variables (i.e., blood count variables) at the 95% confidence level. All statistical analyses were performed with the statistical software Minitab® version 20.4 (MINITAB, 2021).

RESULTS AND DISCUSSION

Table 1 shows the means ± standard error for the WBCC response variable (x 10^9 cells L-1) of the blood count. It was observed that only on day 7 were differences (P<0.05) evidenced between the EGs. Among the EGs, only the EG-Control showed differences between days of the experiment (p<0.05). The rest of the GA showed no differences (P>0.05) between them, nor between the remaining days considered in this study. In the same sense, the RBCC response variable (×10^12 cells L-1) showed differences (P<0.05) only among the ewes included in the EG-eCG14. For the rest of the GA and days, no differences were observed (P>0.05) in the RBCC response variable (Table 2).

Table 1: Mean±standard error of the white blood cell count “leukocytes” (×10^9 cells L-1) in nulliparous sheep, synchronized with two doses of prostaglandin F2α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and a control group in northern Mexico (25oN).



Table 2: Mean ± standard error of the red blood cell count “erythrocytes” (×10^12 cells L-1) in nulliparous sheep, synchronized with two doses of prostaglandin F2α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and a control group in northern Mexico (25oN).


       
In this context, the WBCC response variable (Table 1) increased throughout the study period, which could be related to an immune response to physiological stress (Aiche et al., 2020). The absence of differences (P>0.05) for this response variable was similar to that reported for other oviparous species, such as cows (Ate et al., 2009) and sows (Zvorc et al., 2006). On the contrary, the results are inconsistent with those reported by Greguła‐Kania et al. (2020), who found significant differences in this variable. In the same sense, the differences between WBCC values could be related to a characteristic alteration of the gestation stage, in which maternal and fetal cells are reciprocally recognized by their immune systems, resulting in fetal maintenance capacity (Greguła‐Kania et al. (2020). Additionally, it has been reported that there is a direct relationship between the leukocyte profile, stress and glucocorticoid levels (Preisler et al., 2000). In the same sense, the results presented in Table 1 were higher than the reference values reported (Stayt, 2022). Similarly, they were high compared with those observed in sheep during the last stage of gestation in some sheep breeds in Brazil, Colombia, Nigeria and Turkey (Adeyeye and Ate, 2016; Bezerra et al., 2017; Cihan et al., 2016; Plaza-Cuadrado et al., 2019).
       
Considering the response variable Ht (%), Table 3 presents the mean ± standard error of the hematocrits. In this sense, no differences (P>0.05) were observed between the EG with respect to the number of days included. Only the EG-eCG14 showed differences (P<0.05) between the values determined for the days of analysis. When analyzing the results of the HbC response variable (g dL-1) (Table 4), it is evident that the EGs reported differences (P<0.05) between them for days 14 and 28, as well as the EG-hCG7, for which differences were determined (P<0.05) between the days considered in the study (Table 4).

Table 3: Mean±standard error of the hematocrits (%) in nulliparous sheep, synchronized with two doses of prostaglandin F2α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and a control group in northern Mexico (25oN).



Table 4: Mean±standard error of the hemoglobin concentration (g dL-1) in nulliparous sheep, synchronized with two doses of prostaglandin F2α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and control group in northern Mexico (25oN).


       
In the same sense, Ht levels showed a trend similar to the response variables discussed in the previous paragraphs (Table 3). This is not consistent with what was reported in 2021 by Abdelghany et al., (2021), who compared pregnant and non-pregnant ewes, finding that pregnant ewes showed significant reductions for the RBCC, Ht and MVC variables. Similarly, Aiche et al., (2020) found reductions in the percentage of Ht as gestation progressed in sheep. Likewise, the results obtained are lower than those reported by El-Malky et al., (2019) and Cihan et al., (2016) and also lower than the reference values reported by Stayt in 2022, who reported an optimal range of 27 to 45% Ht in pregnant ewes. However, increases in Ht and Hb concentrations have also been reported in pregnant ewes (Sharma et al., 2015). In addition, Brito et al. (2006) did not observe differences in hematological components between pregnant and non-pregnant ewes, although they reported that Ht decreased as pregnancy progressed.
       
Additionally, with respect to Ht, Greguła‐Kania et al. (2020) observed a dilution of erythrocyte concentration (lower number of erythrocytes, hematocrit and hemoglobin) in pregnant ewes, which is consistent with what was reported in this study (Table 2-4). This could be a physiological response to the decrease in blood viscosity, which improves blood supply to small blood vessels and to the newly formed vascular bed in the uterus and maternal placenta (Greguła‐Kania et al., 2020; Habibu et al., 2017). In the same sense, the RBCC values found in this study were lower than those reported by Aiche et al., (2020), as well as those reported by Bezerra et al. (2017), El-Malky et al. (2019) and Plaza-Cuadrado et al. (2019). Additionally, a study by Soliman (2014) in Ossimi sheep showed that physiological state can lead to significant (p<0.05) modifications in hematological parameters, such as RBCC and Hb. However, Brito et al., (2006) found no variation in hematological parameters between pregnant and lactating ewes when well fed. Additionally, the HbC values found are higher than those reported by El-Malky et al. (2019) in pregnant sheep and Plaza-Cuadrado et al. (2019), but similar to those reported by Adeyeye and Ate (2016) and Bezerra et al. (2017), who recorded higher HbC values in ewes at the end of gestation.
       
Table 5 and 6 present the means ± standard errors for the response variables MCHb (pg) and MCHbC (g dL-1), respectively. Considering this, it was determined that there were no differences (P>0.05) in all interactions between the EGs and the days considered in the study.

Table 5: Mean±standard error of the mean corpuscular hemoglobin (pg) in nulliparous sheep, synchronized with two doses of prostaglandin F2α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and a control group in northern Mexico (25oN).



Table 6: Mean±standard error of the mean corpuscular hemoglobin concentration (g dL-1) in nulliparous sheep, synchronized with two doses of prostaglandin F2α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and control group in northern Mexico (25oN).


       
Regarding RBCC and HbC values, it has been reported that they play an important role in maintaining pregnancy and the survival of the fetus by regulating fetal oxygen supply (Greguła‐Kania et al., 2020). However, the variations evidenced for the RBCC (Table 2) and HbC (Table 5) response variables are likely due to hemodilution resulting from increased plasma volume expansion (Rabea et al., 2024). This plays an important role, particularly in pregnant ewes of twin fetuses, by reducing blood viscosity and thus improving blood flow in the vessels located in the uterus and udder (Karaşahi̇n et al., 2023). In the same sense, the values identified in this study are similar to those reported by Antunović et al. (2011) in Croatia. The absence of significant differences in the response variables RBCC, Ht and MVC was similar to that reported by Ate et al., (2009) in cows, but it contradicts the findings of Zvorc et al. (2006) in pigs, probably due to species differences. Additionally, it has been reported that gestational stress may be responsible for this, given that reproductive status is associated with hormonal changes, particularly steroid hormones, which are known to be immunosuppressive (Adeyeye and Ate, 2016). It has also been suggested that a high rate of red blood cell destruction in mammary cells is responsible for low Ht, along with the mobilization of water to the mammary glands (El-Sherif and Assad, 2001).
       
Continuing with the discussion of the results of the response variables MCHb and MCHbC showed no differences (P>0.05), however, decreases were determined during the period analyzed (Tables 5 and 6). This has been associated with maintaining blood oxygen content (El-Sherif and Assad, 2001; Miglio et al., 2015). From this perspective, the MCHbC values recorded in this study were much higher than those reported by El-Malky et al. (2019), as well as Plaza-Cuadrado et al., (2019) and the reference values reported by Stayt in 2022. On the other hand, the results obtained were consistent with those reported by Bezerra et al. (2017) and Cihan et al. (2016). This could be explained by the fact that the body compensates for the decrease in red blood cells in sheep by increasing the concentration of hemoglobin in erythrocytes, whereas MCHbC increases with advancing pregnancy and decreases after calving (Aiche et al., 2020; Bezerra et al., 2017).
       
The hemogram response variable with the most differences (P<0.05) was MCV (fL), where the EGs differed from day 7, as well as the EGs: ECG7, eCG14 and Control, which showed differences between the days considered in the analysis (Table 7).

Table 7: Mean±standard error of the medium corpuscular volume (fL) in nulliparous sheep, synchronized with two doses of prostaglandin F‚ α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and a control group in northern Mexico (25oN).


       
The value of the MCV (Table 7) obtained in this study was consistent with what was reported by Bezerra et al. (2017), El-Malky et al. (2019) and Plaza-Cuadrado et al. (2019), as well as within the reference range (i.e., 28-40) reported by Stayt in 2022. The determination of these variables (MCHb, MCHbC and MCV) is very important, as they are essential markers of oxygen transport and necessary for cell survival (Aiche et al., 2020). Additionally, it should be noted that an increase in oxygen demand stimulates an adaptive response in which higher hemoglobin concentrations translate into greater oxygen transport (Gravena et al., 2010).
       
Pearson’s correlation analysis was used to evaluate the relationships between the dependent and independent variables considered in the study. In this context, Table 8 presents Pearson’s correlation coefficients and the corresponding significance levels for the relationships among the variables considered. In this regard, low positive correlations of strong to very strong were determined between the variables day and RBCC, HbC and MCHb, day and MCV, WBCC and HbC and HbC and MCV. As well as a very strong moderate positive correlation between the RBCC variable and the MCHbC and MCV variables. Additionally, a high, very strong positive correlation was observed between the variables Ht and MCHb. However, a series of negative correlations was observed, such as the one between the day and Ht variables, which was very low in magnitude and of moderate significance. Another example of negative correlation was determined between the EG and HbC variables, which was low but very strong. Very strong moderate negative correlations were determined between the Ht variable and the MCHbC and MCV variables, as well as between the MCHb and MChbC variables. Finally, a high, very strong negative correlation was observed between the RBCC variable and the Ht and MCHb variables (Table 8).

Table 8: Pearson correlation matrix between groups and response variables analyzed in nulliparous sheep, synchronized with two doses of prostaglandin F2á and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and control group in northern Mexico (25oN).


       
The correlations observed among the different response variables analyzed (Table 8) support the hypothesis of a close relationship among blood components in sheep. This provides valuable information on dynamic changes in hematological parameters during the first month of sheep gestation, which are consistent with the findings of Abdelghany et al., (2021) and Antunović et al. (2013). However, they do not align with others research, such as that reported by Bezerra et al. (2017).
       
Considering the above, the results obtained indicate that the objective of this study was met by evaluating the effects of hCG or eCG administration at 7 or 14 days post-mating on blood counts in early gestation in nulliparous ewes. This is important because it has been reported that analysis of hematological parameters is a reliable method for evaluating the health status of animals, contributing to accurate diagnosis, prognosis and treatment to improve the production and reproduction of sheep (Cetin et al., 2009; Roubies et al., 2006). In this sense, Tables 1 to 7 show the results for various hematological parameters measured at different EGs during the first month post-mating. It could be noted that the variations identified for all hematological response variables in sheep blood during the first month of gestation were within physiological ranges (Table 1-7) (Brooks et al., 2022).

CONCLUSION

Considering that a complete hematological analysis provides reliable information on animal health, it is also an important means of evaluating the production, reproduction and adaptability of animals to different stressful conditions. It was found that the values of the hematological profile of the ewes in the first month of gestation increased for the variables WBCC, RBCC, Ht, HbC and MCV; however, most of the increases were not significant. Highlighting the physiological adjustments developed by the ewes to achieve adequate fetal development, as well as their ability to adapt to the demands of pregnancy. Based on the results obtained, it is recommended to expand the study to determine reference values applicable to the study area and to include additional biochemical parameters. Therefore, future research in this area could deepen understanding of the interrelationships involved to improve the general welfare and reproductive outcomes of sheep.

ACKNOWLEDGEMENT

The authors acknowledge the Antonio Narro Agrarian Autonomous University - Laguna Unit for all the support provided for the completion of this study.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
Informed consent was obtained from the owner of the animals involved in this study. All experimental procedures and animal management used in this study were performed in accordance with international and national standards of ethics, care and animal welfare for research. Additionally, this investigation had institutional approval, with reference number UAAAN-UL-18-3060.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Effect of Treatment with Equine or Human Chorionic Gonadotropin on Blood Count Values in Early Gestation in Nulliparous Sheep in Northern Mexico

    J
    Jesús Armijo-Nájera1
    A
    Ariadna Vanessa Alvarado-Espino2
    F
    Francisco Gerardo Véliz-Deras3,*
    https://orcid.org/0000-0002-5105-1508
    I
    Irene Concepción Chavarría-Neri3
    G
    Gerardo Arellano-Rodriguez3
    J
    Jessica Maria Flores-Salas3
    O
    Oscar Ángel-García3
    C
    Cayetano Navarrete-Molina3
    F
    Francisco Gerardo Véliz-Romero1
    A
    Alan Sebastian Alvarado-Espino3,*
    https://orcid.org/0000-0003-4590-1673
    1Graduate Program-Agricultural and Livestock Sciences, Antonio Narro Agrarian Autonomous University, Laguna Unit, Torreon Coahuila 27054, Mexico.
    2Faculty of Veterinary Medicine and Animal Science Campus 2, Autonomous University of Chiapas, Tuxtla Gutiérrez, Chiapas 29050, Mexico.
    3Regional Division of Animal Science, Antonio Narro Agrarian Autonomous University, Laguna Unit, Torreon, Coahuila 27054, Mexico.

    ABSTRACT

    Background: Hormonal treatments can influence hematological profiles through direct and indirect mechanisms in sheep’s. The aim was to evaluate the effect of administering equine or human chorionic gonadotropin (eCG, hCG, respectively) at 7 or 14 days post-mating on blood count values in early pregnancy of nulliparous ewes.

    Methods: A total of 63 ewes were used, which were randomly assigned to five experimental groups (EG): EG-eCG7 (300 IU of eCG, 7 days post-mating (DPM)), EG-hCG7 (300 IU of hCG 7 DPM), EG-eCG14 (300 IU of eCG 14 DPM), EG-hCG14 (300 IU of hCG 14 DPM) and; EG-Control (without hormonal treatment). Hematologic variables were determined on days 7, 14 and 28 DPM and included white blood cell count (WBCC), red blood cell count (RBCC), hematocrits (Ht), hemoglobin concentration (HbC), mean corpuscular hemoglobin (MCHb), mean corpuscular hemoglobin concentration (MCHbC) and medium corpuscular volume (MCV). An ANOVA and Pearson correlation were performed on the response variables.

    Result: The results showed differences in WBCC on day 7 between the EGs and between days for the EG-Control. The RBCC variable only showed differences between the days of the EG-eCG14. The Ht variable reported differences between the days of the EG-eCG14. Regarding HbC, differences were determined between the EGs for days 14 and 28, as well as between the days of the EG-hCG7. Additionally, positive and negative correlations were evidenced between the variables considered. Based on the above, it is concluded that in the first month of gestation, the variables WBCC, RBCC, Ht, HbC and MCV increase in pregnant ewes. The aforementioned contributes to determining ewes’ reproductive performance and improving their productive and reproductive parameters.

    INTRODUCTION

    One of the main factors affecting the productivity and profitability of sheep herds is reproductive efficiency (Magaña-Monforte et al., 2024). In this context, hormonal synchronization protocols are commonly used to improve reproductive efficiency (Ashour et al., 2018; Murphy, 2012; Sanchez-Ramos et al., 2022). These protocols include the application of equine chorionic gonadotropin (eCG) or human chorionic gonadotropin (hCG) to stimulate follicular development and ovulation (Ashour et al., 2018; Husein and Haddad, 2006; Murphy, 2012). However, hormonal treatments can influence hematological profiles through direct and indirect mechanisms (Bamerny et al., 2022; El-Sayed et al., 2025). This is especially important, as hemograms provide an overview of the physiological and nutritional status of sheep across reproductive stages and facilitate the diagnosis of conditions (Bamerny et al., 2022; Bhat et al., 2017; Mauffré et al., 2016; Porcu et al., 2020; Rabea et al., 2024). In this sense, hematological parameters such as white blood cell count  (WBCC) “leukocytes”, red blood cell count (RBCC) “erythrocytes”, hematocrits (Ht), hemoglobin concentration (HbC), mean corpuscular hemoglobin (MCHb), mean corpuscular hemoglobin concentration (MCHbC) and medium corpuscular volume (MCV) undergo dynamic alterations throughout pregnancy (Rabea et al., 2024). These fluctuations are usually attributed to hemodilution, fetal metabolic demands and changes in plasma volume (Bamerny et al., 2022; Rabea et al., 2024).
           
    Regarding the use of these hormones in hormonal treatments, eCG has a long biological half-life due to glycosylation (Murphy, 2012). And that its application increases twin birth rates, by promoting multiple ovulations through its dual activity, similar to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (Martinez-Ros and Gonzalez-Bulnes, 2019; Porcu et al., 2020). Although eCG is used for reproductive purposes, its interaction with homeostatic mechanisms justifies the monitoring of the number of leukocytes (Bizjak et al., 2018; Gallagher, 2013). This is important, as fluctuations in MCV, MCHb and MCHbC indicate physiological changes during hormonal protocols (Porcu et al., 2020). In this regard, eCG synchronization doses, ranging from 350 IU to 500 IU, have been reported to induce estrus without altering systemic health (Husein and Haddad, 2006; Sanchez-Ramos et al., 2022). In other research, including studies on glycogenic and eCG formulations, results showed that while high doses of other substances affect HbC and MCV, hormonal treatments typically keep rates within physiological limits (Husein and Haddad, 2006; Porcu et al., 2020; Sanchez-Ramos et al., 2022). This suggests that the use of hormonal reproductive protocols with eCG are physiologically compatible with the health of sheep (Dimauro et al., 2008; Martinez-Ros and Gonzalez-Bulnes, 2019; Porcu et al., 2020; Wildeus, 2000).
           
    Considering the use of hCG, in the hormonal protocols of small ruminants, it has been reported that it works as an analogue of LH (Adeyeye and Ate, 2016; Aiche et al., 2020; Khan et al., 2009) and that its main application consists of the synchronization of estrus and the induction of ovulation in sheep and goats (Quintero et al., 2015). In this respect, the application of hCG stimulates the formation of accessory corpus luteus, which improves the endogenous secretion of progesterone (P4) (Adeyeye and Ate, 2016; Khan et al., 2009; Lashari and Tasawar, 2010). Additionally, it has been reported that hCG administration, on day 12 post-mating, aims to increase P4 concentrations to prevent luteolysis (Quintero et al., 2015). This facilitates embryo growth and favors placentation (Cam and Kuran, 2004), correlating with higher pregnancy and fertility rates in various sheep breeds (Quintero et al., 2015). In addition, research on the impact of hCG on the hemogram provides data for the clinical follow-up of sheep under intensive reproductive management (El-Sherif and Assad, 2001; El-Sayed et al., 2025).
           
    Therefore, the correlation between the administration of eCG or hCG and immediate hematological changes in sheep requires additional research to improve animal health monitoring standards (Mattos et al., 2011; Murphy, 2012). These assessments can clarify how these hormones influence systemic variables beyond the hypothalamic-adenopituitary-ovarian axis (Sanchez-Ramos et al., 2022; Wei et al., 2016). In small ruminants (i.e., sheep and goats), metabolic adjustments associated with hormonal mani-pulation and physiological stress can lead to variations in leukocyte and neutrophil counts (Cismaru et al., 2024; Darwish and El-Ebissy, 2019). Therefore, integrating hematological monitoring into routine reproductive management improves the well-being and productivity of sheep herds (Porcu et al., 2020; Wei et al., 2016). Under-standing the relationships between hormone application and hematological monitoring is necessary to refine protocols used in global sheep production (Gãvan et al., 2010; Greguła‐Kania et al., 2020). Therefore, the objective of this study was to evaluate the effect of hCG or eCG administration at 7 or 14 days post-mating on blood count values in early gestation in nulliparous ewes. In order to contribute to improving the reproductive success of sheep, which will improve the income of sheep farming families, while promoting a sustainable vision aligned with the fulfillment of the sustainable development goals of the 2030 Agenda (Autukaitė et al., 2021; Navarrete-Molina et al., 2024; Pampori et al., 2025).

    MATERIALS AND METHODS

    General
     
    All experimental procedures and animal management used in this study were performed in accordance with international and national standards of ethics, care and animal welfare for research (FASS, 2010; NAM, 2002). The period during which the experiment was conducted included the days between September 15 and December 4, 2025. Additionally, this investigation had institutional approval, with reference number UAAAN-UL-18-3060.
     
    Location and environmental conditions of the study area
     
    The sheep production unit, where the study was carried out, is an intensive farm in the Comarca Lagunera (25°37′
    N, 103°16′  W, 1113 meters above sea level) in north-central Mexico. The region is classified as a semi-arid ecosystem, registering an average annual temperature of 23.8oC, with maximums of 41.0oC in summer and minimums of -1.0oC in winter and an average annual rainfall of 230 mm. Additionally, it registers relative humidity ranging from 12% to 61% and photoperiods ranging from 13 h 41 min during the spring solstice (June) to 10 h 19 min during the winter solstice (December) (CONAGUA, 2025; INEGI, 2010).
     
    Animals and experimental design
     
    In this study, 63 Dorper × Blackbelly and Dorper ×  Katahdin crossbred nulliparous ewes, 7 to 9 months old, with a live weight of 32.2±4.4 kg, were used. At the beginning of the experiment and to synchronize the onset of estrus and ovulation, all ewes were administered two doses of prostaglandin F‚ α (75 μg of D-cloprostenol sodium; Estrumate®, Darmstadt, Germany) with an interval of 12 days between applications. After the manifestation of estrus, the ewes were exposed to sexually active Dorper males for seven days to ensure mating. For this, the males were marked with vegetable paint in the sternal region to easily identify the mounted females. The day on which the painted sheep of the lumbar region was observed for the first time was considered the day of mating.
           
    Based on the above, the ewes were randomly assigned to five experimental groups (EG): EG-eCG7 (n = 14), the ewes in this group received 300 IU of eCG (Folligon®, Darmstadt, Germany) intramuscularly (IM) 7 days post-mating; EG-hCG7 (n = 13) females included in this group received 300 IU via hCG IM (Chorulon®, Darmstadt, Germany) 7 days post-mating; EG-eCG14 (n = 13) received 300 IU of eCG via IM 14 days post-mating; EG-hCG14 (n = 13) received 300 IU via IM of hCG 14 days post-mating and; EG-Control (n = 10) who did not receive post-mating hormonal treatment. In addition, on days 7 and 14 post-mating, all ewes that did not receive hormonal treatment received 1.5 mL of sterile injectable solution (Isosol MDKR, Mediker, Gomez Palacio, Durango, Mexico). The sheep were fed a diet based on food residues from a highly industrialized dairy cattle production system twice a day (1000 and 1800 h). Likewise, all females had ad libitum access to drinking water, mineral salts and shade. To guarantee the physiological status of the females (i.e. pregnant), a diagnosis of pregnancy was made at 32 days post-mating by transrectal ultrasono-graphy, which was confirmed by visualization of intrauterine fluid (embryonic vesicle) and the presence of echogenic structures within anechoic cavities compatible with viable embryos, for which an ultrasound (Chison ECO5 Doppler®, Wuxi, Jiangsu, China) and complemented with a rectal transducer (7.5 MHz, Color® Doppler, Wuxi, Jiangsu, China) was used, confirming that 100% of the experimental units that were included in the EGs were pregnant.
     
    Blood sampling and quantification of the values of the blood count to be evaluated
     
    On days 7, 14 and 28 post-mating, blood samples were taken from all ewes (n = 63) to determine blood count values. To this end, at 08:00 h on an empty stomach, samples of 5 mL of blood were obtained by jugular venipuncture with BD Vacutainer® tubes of 9 mL (Franklin Lakes, New Jersey, USA). The tubes contained 7.2 mg of dehydrated K2EDTA in their inner walls. This compound acts as an anticoagulant by binding calcium ions and interrupting the blood-clotting process. To continue the analysis of the samples, the hemogram variables considered in the study were quantified. For this, an automated hematological analyzer Hemalyzer HVET 2000 brand HLab (Denver, Colorado, USA), was used. All procedures were performed by a trained technician. The blood response variables considered in the study included: white blood cell count "leukocytes" (WBCC, ×10^9 cells L-1); red blood cell count "erythrocytes" (RBCC, ×10^12 cells L-1); hematocrits (Ht, %); hemoglobin concentration (HbC, g dL-1); mean corpuscular hemoglobin (MCHb, pg); mean corpuscular hemoglobin concentration (MCHbC, g dL-1) and medium corpuscular volume (MCV, fL). All analyses were performed in the Bromatology Laboratory of the Antonio Narro Agrarian Autonomous University, Laguna Unit.
     
    Statistical analysis
     
    The Kolmogorov-Smirnov test at a confidence level of 95% was used to verify the normality of the data. Considering that the response variables RBCC, Ht, MCHbC and MCV did not show a normal distribution, a Box-Cox transformation was performed. Equality of variances was assessed using Levene’s test with a 95 % confidence level. Subsequently, an analysis of variance was performed, using Fisher’s LDS method to compare means, considering a 95% confidence level. Additionally, the mean and standard error were calculated for each response variable. Finally, Pearson’s correlation coefficient was computed between the independent variables (i.e., experimental group and day) and the dependent variables (i.e., blood count variables) at the 95% confidence level. All statistical analyses were performed with the statistical software Minitab® version 20.4 (MINITAB, 2021).

    RESULTS AND DISCUSSION

    Table 1 shows the means ± standard error for the WBCC response variable (x 10^9 cells L-1) of the blood count. It was observed that only on day 7 were differences (P<0.05) evidenced between the EGs. Among the EGs, only the EG-Control showed differences between days of the experiment (p<0.05). The rest of the GA showed no differences (P>0.05) between them, nor between the remaining days considered in this study. In the same sense, the RBCC response variable (×10^12 cells L-1) showed differences (P<0.05) only among the ewes included in the EG-eCG14. For the rest of the GA and days, no differences were observed (P>0.05) in the RBCC response variable (Table 2).

    Table 1: Mean±standard error of the white blood cell count “leukocytes” (×10^9 cells L-1) in nulliparous sheep, synchronized with two doses of prostaglandin F2α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and a control group in northern Mexico (25oN).



    Table 2: Mean ± standard error of the red blood cell count “erythrocytes” (×10^12 cells L-1) in nulliparous sheep, synchronized with two doses of prostaglandin F2α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and a control group in northern Mexico (25oN).


           
    In this context, the WBCC response variable (Table 1) increased throughout the study period, which could be related to an immune response to physiological stress (Aiche et al., 2020). The absence of differences (P>0.05) for this response variable was similar to that reported for other oviparous species, such as cows (Ate et al., 2009) and sows (Zvorc et al., 2006). On the contrary, the results are inconsistent with those reported by Greguła‐Kania et al. (2020), who found significant differences in this variable. In the same sense, the differences between WBCC values could be related to a characteristic alteration of the gestation stage, in which maternal and fetal cells are reciprocally recognized by their immune systems, resulting in fetal maintenance capacity (Greguła‐Kania et al. (2020). Additionally, it has been reported that there is a direct relationship between the leukocyte profile, stress and glucocorticoid levels (Preisler et al., 2000). In the same sense, the results presented in Table 1 were higher than the reference values reported (Stayt, 2022). Similarly, they were high compared with those observed in sheep during the last stage of gestation in some sheep breeds in Brazil, Colombia, Nigeria and Turkey (Adeyeye and Ate, 2016; Bezerra et al., 2017; Cihan et al., 2016; Plaza-Cuadrado et al., 2019).
           
    Considering the response variable Ht (%), Table 3 presents the mean ± standard error of the hematocrits. In this sense, no differences (P>0.05) were observed between the EG with respect to the number of days included. Only the EG-eCG14 showed differences (P<0.05) between the values determined for the days of analysis. When analyzing the results of the HbC response variable (g dL-1) (Table 4), it is evident that the EGs reported differences (P<0.05) between them for days 14 and 28, as well as the EG-hCG7, for which differences were determined (P<0.05) between the days considered in the study (Table 4).

    Table 3: Mean±standard error of the hematocrits (%) in nulliparous sheep, synchronized with two doses of prostaglandin F2α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and a control group in northern Mexico (25oN).



    Table 4: Mean±standard error of the hemoglobin concentration (g dL-1) in nulliparous sheep, synchronized with two doses of prostaglandin F2α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and control group in northern Mexico (25oN).


           
    In the same sense, Ht levels showed a trend similar to the response variables discussed in the previous paragraphs (Table 3). This is not consistent with what was reported in 2021 by Abdelghany et al., (2021), who compared pregnant and non-pregnant ewes, finding that pregnant ewes showed significant reductions for the RBCC, Ht and MVC variables. Similarly, Aiche et al., (2020) found reductions in the percentage of Ht as gestation progressed in sheep. Likewise, the results obtained are lower than those reported by El-Malky et al., (2019) and Cihan et al., (2016) and also lower than the reference values reported by Stayt in 2022, who reported an optimal range of 27 to 45% Ht in pregnant ewes. However, increases in Ht and Hb concentrations have also been reported in pregnant ewes (Sharma et al., 2015). In addition, Brito et al. (2006) did not observe differences in hematological components between pregnant and non-pregnant ewes, although they reported that Ht decreased as pregnancy progressed.
           
    Additionally, with respect to Ht, Greguła‐Kania et al. (2020) observed a dilution of erythrocyte concentration (lower number of erythrocytes, hematocrit and hemoglobin) in pregnant ewes, which is consistent with what was reported in this study (Table 2-4). This could be a physiological response to the decrease in blood viscosity, which improves blood supply to small blood vessels and to the newly formed vascular bed in the uterus and maternal placenta (Greguła‐Kania et al., 2020; Habibu et al., 2017). In the same sense, the RBCC values found in this study were lower than those reported by Aiche et al., (2020), as well as those reported by Bezerra et al. (2017), El-Malky et al. (2019) and Plaza-Cuadrado et al. (2019). Additionally, a study by Soliman (2014) in Ossimi sheep showed that physiological state can lead to significant (p<0.05) modifications in hematological parameters, such as RBCC and Hb. However, Brito et al., (2006) found no variation in hematological parameters between pregnant and lactating ewes when well fed. Additionally, the HbC values found are higher than those reported by El-Malky et al. (2019) in pregnant sheep and Plaza-Cuadrado et al. (2019), but similar to those reported by Adeyeye and Ate (2016) and Bezerra et al. (2017), who recorded higher HbC values in ewes at the end of gestation.
           
    Table 5 and 6 present the means ± standard errors for the response variables MCHb (pg) and MCHbC (g dL-1), respectively. Considering this, it was determined that there were no differences (P>0.05) in all interactions between the EGs and the days considered in the study.

    Table 5: Mean±standard error of the mean corpuscular hemoglobin (pg) in nulliparous sheep, synchronized with two doses of prostaglandin F2α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and a control group in northern Mexico (25oN).



    Table 6: Mean±standard error of the mean corpuscular hemoglobin concentration (g dL-1) in nulliparous sheep, synchronized with two doses of prostaglandin F2α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and control group in northern Mexico (25oN).


           
    Regarding RBCC and HbC values, it has been reported that they play an important role in maintaining pregnancy and the survival of the fetus by regulating fetal oxygen supply (Greguła‐Kania et al., 2020). However, the variations evidenced for the RBCC (Table 2) and HbC (Table 5) response variables are likely due to hemodilution resulting from increased plasma volume expansion (Rabea et al., 2024). This plays an important role, particularly in pregnant ewes of twin fetuses, by reducing blood viscosity and thus improving blood flow in the vessels located in the uterus and udder (Karaşahi̇n et al., 2023). In the same sense, the values identified in this study are similar to those reported by Antunović et al. (2011) in Croatia. The absence of significant differences in the response variables RBCC, Ht and MVC was similar to that reported by Ate et al., (2009) in cows, but it contradicts the findings of Zvorc et al. (2006) in pigs, probably due to species differences. Additionally, it has been reported that gestational stress may be responsible for this, given that reproductive status is associated with hormonal changes, particularly steroid hormones, which are known to be immunosuppressive (Adeyeye and Ate, 2016). It has also been suggested that a high rate of red blood cell destruction in mammary cells is responsible for low Ht, along with the mobilization of water to the mammary glands (El-Sherif and Assad, 2001).
           
    Continuing with the discussion of the results of the response variables MCHb and MCHbC showed no differences (P>0.05), however, decreases were determined during the period analyzed (Tables 5 and 6). This has been associated with maintaining blood oxygen content (El-Sherif and Assad, 2001; Miglio et al., 2015). From this perspective, the MCHbC values recorded in this study were much higher than those reported by El-Malky et al. (2019), as well as Plaza-Cuadrado et al., (2019) and the reference values reported by Stayt in 2022. On the other hand, the results obtained were consistent with those reported by Bezerra et al. (2017) and Cihan et al. (2016). This could be explained by the fact that the body compensates for the decrease in red blood cells in sheep by increasing the concentration of hemoglobin in erythrocytes, whereas MCHbC increases with advancing pregnancy and decreases after calving (Aiche et al., 2020; Bezerra et al., 2017).
           
    The hemogram response variable with the most differences (P<0.05) was MCV (fL), where the EGs differed from day 7, as well as the EGs: ECG7, eCG14 and Control, which showed differences between the days considered in the analysis (Table 7).

    Table 7: Mean±standard error of the medium corpuscular volume (fL) in nulliparous sheep, synchronized with two doses of prostaglandin F‚ α and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and a control group in northern Mexico (25oN).


           
    The value of the MCV (Table 7) obtained in this study was consistent with what was reported by Bezerra et al. (2017), El-Malky et al. (2019) and Plaza-Cuadrado et al. (2019), as well as within the reference range (i.e., 28-40) reported by Stayt in 2022. The determination of these variables (MCHb, MCHbC and MCV) is very important, as they are essential markers of oxygen transport and necessary for cell survival (Aiche et al., 2020). Additionally, it should be noted that an increase in oxygen demand stimulates an adaptive response in which higher hemoglobin concentrations translate into greater oxygen transport (Gravena et al., 2010).
           
    Pearson’s correlation analysis was used to evaluate the relationships between the dependent and independent variables considered in the study. In this context, Table 8 presents Pearson’s correlation coefficients and the corresponding significance levels for the relationships among the variables considered. In this regard, low positive correlations of strong to very strong were determined between the variables day and RBCC, HbC and MCHb, day and MCV, WBCC and HbC and HbC and MCV. As well as a very strong moderate positive correlation between the RBCC variable and the MCHbC and MCV variables. Additionally, a high, very strong positive correlation was observed between the variables Ht and MCHb. However, a series of negative correlations was observed, such as the one between the day and Ht variables, which was very low in magnitude and of moderate significance. Another example of negative correlation was determined between the EG and HbC variables, which was low but very strong. Very strong moderate negative correlations were determined between the Ht variable and the MCHbC and MCV variables, as well as between the MCHb and MChbC variables. Finally, a high, very strong negative correlation was observed between the RBCC variable and the Ht and MCHb variables (Table 8).

    Table 8: Pearson correlation matrix between groups and response variables analyzed in nulliparous sheep, synchronized with two doses of prostaglandin F2á and treated with 300 IU of equine or human chorionic gonadotropin administered on day 7 or 14 post-mating and control group in northern Mexico (25oN).


           
    The correlations observed among the different response variables analyzed (Table 8) support the hypothesis of a close relationship among blood components in sheep. This provides valuable information on dynamic changes in hematological parameters during the first month of sheep gestation, which are consistent with the findings of Abdelghany et al., (2021) and Antunović et al. (2013). However, they do not align with others research, such as that reported by Bezerra et al. (2017).
           
    Considering the above, the results obtained indicate that the objective of this study was met by evaluating the effects of hCG or eCG administration at 7 or 14 days post-mating on blood counts in early gestation in nulliparous ewes. This is important because it has been reported that analysis of hematological parameters is a reliable method for evaluating the health status of animals, contributing to accurate diagnosis, prognosis and treatment to improve the production and reproduction of sheep (Cetin et al., 2009; Roubies et al., 2006). In this sense, Tables 1 to 7 show the results for various hematological parameters measured at different EGs during the first month post-mating. It could be noted that the variations identified for all hematological response variables in sheep blood during the first month of gestation were within physiological ranges (Table 1-7) (Brooks et al., 2022).

    CONCLUSION

    Considering that a complete hematological analysis provides reliable information on animal health, it is also an important means of evaluating the production, reproduction and adaptability of animals to different stressful conditions. It was found that the values of the hematological profile of the ewes in the first month of gestation increased for the variables WBCC, RBCC, Ht, HbC and MCV; however, most of the increases were not significant. Highlighting the physiological adjustments developed by the ewes to achieve adequate fetal development, as well as their ability to adapt to the demands of pregnancy. Based on the results obtained, it is recommended to expand the study to determine reference values applicable to the study area and to include additional biochemical parameters. Therefore, future research in this area could deepen understanding of the interrelationships involved to improve the general welfare and reproductive outcomes of sheep.

    ACKNOWLEDGEMENT

    The authors acknowledge the Antonio Narro Agrarian Autonomous University - Laguna Unit for all the support provided for the completion of this study.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    Informed consent was obtained from the owner of the animals involved in this study. All experimental procedures and animal management used in this study were performed in accordance with international and national standards of ethics, care and animal welfare for research. Additionally, this investigation had institutional approval, with reference number UAAAN-UL-18-3060.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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