Buffalos (Bubalus bubalis) are a common species in the Middle East (1) and Africa (2), and have great potential due to their critical role in farming and agriculture in those developing countries (3–5). One of the major restraints in the utilization of domestic buffalo reproductive capacity as compared to cattle has been its characteristically poorer reproductive functionality and efficiency (6) that is associated with silent heat, missing many behavioral signs, and lesser conception rates (7, 8). So, studying a basic reproductive pattern is of great importance for enhancing the reproductive efficiency of buffalos (9). The corpus luteum (CL) is the primary reproductive gland responsible for progesterone production, and is required for the establishment and progression of the gestation period; moreover, CL plays a critical role in many reproductive processes, such as successive implantation and embryonic development (10, 11). Histologically, CL consists of cells with a steroidogenic and non-steroidogenic nature (12). The steroidogenic cells, which are responsible for progesterone production, are composed of luteal cells (large and small cells that originate from both granulosa and theca cells), while the non-steroidogenic cells are composed of fibroblasts, endothelial cells, and macrophage (13, 14). Although luteal functions have been evaluated in many studies (15, 16), many specific regulatory factors related to luteal vascularization need future studies, especially in buffalos. The size of the CL at the mid-luteal phase of the estrous cycle ranged from 1 to 2 cm compared to that at pregnancy which ranged from 2 to 2.5 cm (17). Generally, the CL in buffalo is smaller compared to that in cattle (18) and associated with lower progesterone levels, which in turn may affect embryonic mortality (19, 20) and adversely impact the maintenance of pregnancy (21, 22). Some studies have reported that the adequate development of the CL in association with progesterone levels (P4) is needed to prevent embryonic mortality (23); in addition, pregnant buffalos reported a greater diameter of CL linked to a marked linear increase in plasma P4 levels that positively affects the luteal vascularity (LV) (24). Another study has reported a higher average timed velocity (TAV cm/sec) of the luteal artery in pregnant buffalos compared to non-pregnant buffalos (25). Moreover, lower pregnancy rates are observed with lower CL vascularization after 5 days of mating (26). The luteal and early normal pregnancy phases are associated with marked changes in the cardiovascular hemodynamic system, such as elevated blood flow volume and decreased Doppler indices, especially vascular resistance index (27, 28), as all these changes are followed by increased levels of nitric oxide (NO) and its metabolites (NOMs) in the form of nitrite and nitrate, which lead to the improved response of smooth muscle on NO reaction (29). Little is known about the histological structure of CL, using the Doppler technique that is based on the anatomical determination of the luteal artery, as well as characterization of each luteal stage in normal luteal and pregnant phases in Egyptian domestic buffalo. Therefore, this current study aimed to compare the complete growth and development of CL in domestic buffalos from day 5 until day 40 after ovulation either in pregnant or non-pregnant buffalo, and whether luteal vascularity (LV) with progesterone (P4) and nitric oxide (NO) could determine luteal functionality or not.

The current study compares the complete growth and development of CL in domestic buffalos from day 5 until day 40 after ovulation. CL plays a critical role in the gestation maintenance and establishment in many species because of its ability to produce an adequate P4 level (10, 45), therefore, any decline of this mechanism could affect embryonic mortality as previously demonstrated in cows (46). This study shows a significant elevation in CL area from day 20 until day 40 in pregnant buffalos compared to non-pregnant buffalos with a marked elevation of plasma P4 levels in the same group, which could help determine the importance of adequate CL diameter and area to establish the first early stage of pregnancy (47). Additionally, some studies reported a higher CL area in early pregnant females compared to non-pregnant ones (25), but others did not show a significant difference between the two groups (48). Similarly, some studies reported an elevation in plasma P4 levels from day 7 to day 10 after mating in pregnant females (25, 47) as high P4 levels were responsible mainly for greater embryonic development (23). In addition, the P4 levels remained elevated in some studies until 6–8 months of pregnancy (49).

Increases in CL diameter and progesterone level were greater in pregnant than in non-pregnant buffalos, indicating the importance of adequate CL size for maintenance of pregnancy (47). Lesser plasma progesterone levels from day 10 to 20 after AI in buffalos were associated with the death of an embryo (22), demonstrating the pivotal role of CL for pregnancy stage development and maintenance. The variation in the serum P4 concentrations throughout the estrous cycle of non-pregnant and pregnant buffalos is primarily dependent on the blood flow, CL area, amount of steroidogenic tissue (number and size of luteal cells), and its capacity to synthesize progesterone (10), in addition to its lipid contents (50). The development, maintenance, and regression of CL involved remarkable morphological and functional changes during the estrous cycle (51–53). The current study showed that CL had steroidogenic cells involving LLCs and SLCs with morphological characteristics that were reported by Baithalu et al. (14) in buffalo and Ozen et al. (54) and Xavier et al. (55) in a cow. The presence of large lightly stained nuclei of LLCs throughout stages I, II, and III may indicate the activity and hypersecretory LLCs during these phases of the cycle and pregnancy, while luteal cells of Stage IV revealed small more dense nuclei, which is an indication of luteolysis. Furthermore, the presence of vacuolated cytoplasm of LLCs was caused by their lipid contents that vary throughout the estrous cycle and appear in a close association with the synthesis and secretion of progesterone. Kapoor et al. (50) demonstrated that the variations in lipid distribution within the cyclic and regressed CL were inversely associated with the activity of the 3β-hydroxysteroid dehydrogenase enzyme involved in the synthesis of steroid hormones. Our results exhibited lutein cells of stage IV with highly vacuolated cytoplasm. Increasing accumulation of lipids in the regressed luteal cells with a significant decline in production of P4 hormone might be due to degenerated smooth endoplasmic reticulum resulting in absence of 3β-HSD in addition to degenerated mitochondria containing P450 side-chain cleavage (50, 56). Both 3β-HSD and P450 side-chain cleavage were involved in the biosynthesis of progesterone (50). Additionally, during the estrus cycle, the luteal cell population and the size of luteal cells increased and then regressed at stage IV. These results were consistent with references (14, 57, 58). Therefore, these findings reflect the variation in the serum P4 concentrations throughout the estrous cycle of non-pregnant and pregnant buffalos.

Hence, the present study was designed to study the cellular composition of the mature buffalo CL with its functional characterization in relation to progesterone secretory ability and nitric oxide during the normal luteal and pregnancy phases. In the normal luteal phase the level of NOMs was critical as it was related to the luteal vascularization and functionality (59), as after ovulation nitric oxide affects oocyte activation by regulating the calcium channel during the process of fertilization (60), while in the normal early pregnant stage, nitric oxide contributes to the elevation of maternal blood and reduction of blood pressure (61). Moreover, nitric oxide was extremely important in the embryo's multiple divisions (62) as some studies showed that embryonic growth was delayed due to the presence of inhibitory mediators in the blastocyst stage that adversely affects nitric oxide levels (63). Besides nitric oxide, some mediators have been concerned with this phenomenon, such as estradiol and prostacyclin (64).

The elevation in uterine blood flow that was expressed by red and blue colored areas in the pregnant group compared to non-pregnant buffalos could be associated with the buffalo maternal pregnancy recognition that began once the early embryo moved in the fallopian tube and then entered the uterus after 5–6 days post-mating, as the uterine vascularization is included in the implantation procedures expressed by the growth and development of new blood vessels (65, 66). Furthermore, increased serum levels of NOMs in early pregnancy were very important in the vasodilator mechanisms as nitric oxide is shared in the blood pressure regulations and coronary artery vasomotion, therefore any abnormalities in nitric oxide and its metabolites levels could adversely affect pregnancy (67) in form of hypertension and angiogenesis problems (68).

Besides progesterone's important function, CL functionality could also be assessed by luteal vascularization (36). In this study, the CL vascularization was determined using both Doppler indices and PSV (cm/s), as both Doppler indices decreased in pregnant females compared to those in non-pregnant females, while the luteal artery PSV was elevated in the same pregnant group, this could be explained by the inverse relationship between both Doppler indices and Doppler velocities with time average point (TAV) as previously reported in other studies (69–72), in addition, an inverse relationship was observed between blood flow rate and Doppler indices especially PI (73–75). In accordance with our finding, a study reported an increase in both peak and time average velocities (PSV and TAV cm/s) on day 7 after time artificial insemination in pregnant cows (76), as the determination of luteal blood flow total area with Doppler velocities could increase the accuracy of pregnancy prediction (26). Consistent with our Doppler measurements, cyclic CL (stage I, II, and III) and CL of pregnancy were characterized by high vascularization, and most lutein cells were adjacent to engorged blood capillaries that were in harmony with Xavier et al. (55) in the pregnant cow. These results might be due to the high metabolic demand for CL. Moreover, the growth and maintenance of CL and its adequate endocrine function were mainly associated with increasing luteal vascularization and angiogenesis (77, 78). Many studies found a similar positive correlation between CL vascularization in its blood flow and plasma P4 levels after ovulation (79). However, during luteal regression, all lutein cells decreased in their number and shrunk until they disappeared, leaving arteriole-like remnants of blood vessels with an onion-skin arrangement of surrounding myofibroblast and heavily condensed and rounded endothelial cells with dense connective tissue in the residual CL. This finding was in accordance with observations reported by Augustin et al. (80). Increasing thickness of the CT capsule, CT septae, and blood vessels led to reduced blood flow, which in turn resulted in decreased progesterone secretion as observed in the current study.

Moreover, this study revealed the presence of non-steroidogenic cells, mainly fibroblast and endothelial cells. In stages III and IV, the number of fibroblasts notably increased, which was consistent with the results of Baithalu et al. (14) as the fibroblasts were responsible for the synthesis of connective tissue fibers and extracellular matrix (81), and collagen fibers were required to alternate the degenerated and regressed luteal cells. Baithalu et al. (14) reported that, during the late luteal stage, the greater number of macrophages and fibroblasts could serve as a cellular marker of luteal regression. Furthermore, our results revealed a high rate of deposition of collagen fibers in stages III and IV which comes in accordance with Jaglan et al. (53), who observed changes in the collagen concentration with the development and regression of cyclic buffalo CL during the estrous cycle. Iwahashi et al. (82) showed that alterations in the synthesis and distribution of collagen played a primary role in determining the CL structure and function.

The histological structure of CL and assessment of its hemodynamics depending on anatomical identifications could be used extensively to get useful data about CL functional status in both luteal and early pregnant phases. Finally, the evaluation of the luteal artery could be extremely helpful as the artery showed a wave pattern to determine the potential benefits of colored and pulsed Doppler in CL vascularization assessment.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by Faculty of Veterinary Medicine, Cairo University.

EA, IE, YA, AT, NY, and SD designed the protocol and collected the samples. NY worked on the histological examinations. EA and IE performed Doppler and ultrasonographical images, while YA, AT, and SD performed the anatomical vascular architecture. All authors drafted the manuscript, reviewed it, and approved the last version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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