The study was conducted from September to December 2019 at the Department of Veterinary Anatomy and Physiology, University of Nairobi, Kenya. The average temperature was 24°C, and the relative humidity was generally greater than 58%.

The seeds of Moringa oleifera (Lam) aged 2 to 3 years were collected from Masii village of Machakos County in Kenya. The plant was taxonomically identified at the Department of Biology and Biotechnology, University of Nairobi, Kenya, and a voucher specimen (MK032) was deposited in the herbarium at the School of Biological Sciences, University of Nairobi, Chiromo Campus. The seeds were air-dried at 21 to 25°C for 14 days. After spreading, the plant material was ground using an electric mill and the fine powder was collected and stored in airtight glassware until aqueous extraction.

The aqueous extract was obtained using the method described by Okumu et al. (25). Briefly, a total of 1,000 g of dried Moringa oleifera seed powder were aliquoted into a vial wrapped in aluminum foil. Ten l of distilled water was gradually added to the powder with gentle stirring using a magnetic wand until a slurry of uniform consistency was formed. The formed slurry was further stirred using a magnetic stirrer operating at 200 RPM for 48 h. The resulting slurry was centrifuged at 3,000 RPM for 5 min, and the filter media were collected for further processing.

The bioactive compounds were detected using phytochemical tests according to standard methods developed by Harborne (26). For quantitative analysis, the concentration of different compounds mainly alkaloids, saponins, flavonoids, total phenols, glucosides, terpenoids, and tannins was evaluated following the methods developed, respectively, by Harborne (27), Ejikeme et al. (28), Edeoga et al. (29), Amadi et al. (30), Ferguson (31), and Van-Burden and Robinson (32). Table 1 shows the bioactive compounds detected in Moringa oleifera seed aqueous extract.

A total of 28 female rabbits, aged 6 months and weighing 2,286.1 ± 63.5 g (Mean ± SE), purchased from a local recognized and licensed breeder, were used. Throughout the experimental period, feed and water were provided ad libitum to animals. All animals used for the experiment were provided with the basal commercial pelleted ration containing 18.18% crude protein, 13.43% crude fiber, 2,656 MJ/kg diet digestible energy, and 2.29% ether extract that met all nutritional requirements of rabbit does according to the National Research Council (33). The rabbit cages were routinely cleaned every morning before clinical observations to the animals.

During the acclimatization period, the rabbits were housed in wire cages (0.8 × 0.6 × 0.6 m) at room temperature of 21 ± 3°C with animal house relative humidity of 66 ± 3% and kept under the same hygienic and managerial conditions. Before starting the experiment, the animals were weighed, randomly assigned to four groups of seven female rabbits each with comparable body weight, and distributed to T0 and T3 (normal and positive control, respectively) and T1 and T2 (low and high doses of Moringa oleifera, respectively). Thereafter, during 80 consecutive days, animals were submitted to different room temperatures, relative humidity, temperature humidity index (THI), and of Moringa oleifera aqueous seed extract (MOASE) as follows: T0: ambient temperature (18–24°C), 59 ± 0.48%, 23.6 ± 1.52; T1: 35–36°C, 64 ± 0.6%, 32.5 ± 0.7, 100 mg/kg b.w of MOASE; T2: 35–36°C, 64 ± 0.6%, 32.5 ±0.7, 200 mg/kg b.w of MOASE; T3: 35–36°C, 64 ± 0.6%, 32.5 ± 0.7. The heat was induced in each rabbit cage, using electrical heaters (brand: ARMCO from India) from 08:00 h to 16:00 h followed by exposure to the normal air temperature as in the control group from 16.00 h to 08:00 h. During the experimental period, the relative humidity and ambient temperature were recorded twice daily using an automatic thermo-hygrometer (Brand: RC dalys, Size: 48^*28.6^*15.2 cm, temperature precision: ±1°C, hygrometry precision: ±5). The selected range of temperature, relative humidity, and THI was chosen according to the results observed in our previous study (34) and was classified as very severe heat stress (35). Moringa oleifera aqueous seed extracts (MOASE) were administrated per os once a day for 80 days using an endogastric cannula, while normal and negative control animals (T0 and T3, respectively) orally received 10 ml of distilled water daily. 2 weeks after submitting animals to heat stress and gavaging Moringa oleifera aqueous seed extract, each female rabbit was transferred in a single proven fertile males' cage for mating. 10 days after mating, pregnancy was evaluated by abdominal palpation by hands feeling around until finding of little lumps that can pass through fingers as gently probe and search (35). The females failing to conceive were immediately returned to the same buck for another service.

In pregnant and lactating female rabbits, feed intake (F.I.) and weight gain (W.G.) were measured daily and daily body gain was calculated for each female rabbit according to the method described by Sabah and Dalal (9).

The gestation length was obtained by calculating the duration (in days) between the date of the coupling and the date of farrowing (36, 37). The conception rate was estimated as number of services per conception, and the type of mating observed was classified as voluntary mating (i.e., mating receptive does) and hand mating (i.e., mating unreceptive does) (35). The litter size was obtained by counting the number of young rabbits of each female at farrowing and weekly until weaning (36–38). The stillbirth rate was evaluated as the number of stillborn young rabbits on the litter size at birth (38), and the survival rate was calculated as the litter size at weaning on the litter size at birth (37, 38). The young rabbit weight evolution was obtained by weighing young rabbits at birth and every week until 5 weeks post-partum (weaning) (36–38).

For each female, milk yield was evaluated as the difference in weight of the kids before and after suckling. Milk intake per kit was calculated as the milk yield on the number of kits in the litter for each female rabbit, and the efficiency of converting milk into body weight gain was calculated as the kit weight gain on the milk intake per kit (35).

The litter weight was evaluated by weighing the whole kids of the litter, the kit body weight was estimated by weighing separately each kid, and the kit body weight gain was evaluated as the difference between the actual kit weight and the kit weight of the previous week (35). These parameters were estimated at weekly intervals between birth and 35 days of age (weaning).

At the end of the experimental period (80 days), all animals were fasted for 24 h and humanely sacrificed by euthanizing using ether vapor and then dissected. Ten ml (10 ml) of the blood was collected directly by cardiac puncture before euthanasia using ether vapor by inhalation. The obtained blood was then centrifuged, and the resultant serum was kept at −20°C for hormones assessment. After sacrifice, the ovaries were collected by dissection, freed of adipose tissue, washed using saline solution, and blot-dried for histology.

The principal hormones analyzed were as follows: estradiol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone, prolactin (PL), and cortisol. Cortisol was estimated by the radioimmunoassay (RIA) technique using coated tubes kits (Diagnostic Systems Laboratories, Inc. Webster, Texas 77598-4217, USA) and the tracer labeled with iodine-125 (I¹²⁵). Quantitative determination of serum estradiol, FSH, LH, progesterone, and PL was performed using the solid-phase enzyme-linked immunosorbent assay (ELISA) as described by the commercial kit Omega Diagnostic (23961 Craftsman Road, Suite D/E/F, Calabasas, CA 91302). Briefly, 100 uL of samples was added to the appropriate well in the antibody pre-coated Microtiter Plate. Then, 100 uL of PBS (pH 7.0–7.2) was added in the blank control well; 10 uL of Balance Solution was dispensed into 100 uL specimens and then mixed well; 50 uL of the conjugate was added to each well-except the blank control well and then mixed before incubation for 1 h at 37°C. After incubation, the plate was washed using manual washing. The incubation mixture was removed by aspirating contents of the plate into a sink; then, the plate was washed five times with a wash solution. After washing, invert the plate and blot dry by hitting the plate onto absorbent paper until no moisture appears. About 50 uL of Substrate A and 50 uL of Substrate B were added into each well-including blank control well, subsequently, and then incubated for 10–15 min at 20–25°C. After incubation, 50 uL of Stop Solution was added to each well-including blank control well. Therefore, the optical density (O.D.) was determined at 450 nm using a microplate reader immediately. The standard curve was used to determine the concentration of samples as corresponding to the mean absorbance from the standard curve.

All analyses were performed following the manufacturers' recommendations.

Immediately after sacrifice, the left ovary of each female rabbit was removed, fixed in 10.00% formalin for 1 week, then washed, dehydrated with ascending grades of alcohol bath, clarified in xylene immersion, embedded in paraffin, cut at 5.00-μm thickness, and stained with hematoxylin and eosin. The sections were observed under a light microscope (Leica DM 750, X10, and X40) equipped with a DCM35 digital camera (350 Kpixels, USB 2.0), which was connected to the central processing unit of a computer. The images were directly observed on the computer equipped with an image capture program. The captured images were magnified 100 times (100X) for histology and cellular integrity.

Data were compiled into a database using Excel package. For the analysis of the different treatments, the effects among experimental groups and control were assessed using one-way ANOVA. The differences in mean values were compared using the Tukey HSD test at P < 0.05. Data analysis was performed using XLSTAT for Windows 10 Software. The results are expressed as the means ± SD, graphs, and charts. The effect rate (ER) was calculated as the ratio between the value of the control group and the value of the group with the most relevant impact.

The results on feed consumption (Figure 1) showed decrease of 21 and 34%, respectively, in pregnant (A) and lactating (B) female rabbits submitted to heat stress compared to those of the control group. However, in Moringa oleifera aqueous seed extract-treated animals, this parameter increased in a dose-dependent manner. Similar results have been reported in body weight of both pregnant (C) and lactating (D) female rabbits by 8 and 26%, respectively, except at the 5th week where body weight of animals submitted to heat stress and treated with 200 mg/kg b.w. of Moringa oleifera aqueous seed extract. In these animals, body weight was slightly higher by 4% compared to that of the control group in pregnant females (C) and of animals submitted to heat stress and treated with 100 mg/kg b.w. of Moringa oleifera aqueous seed extract which suddenly decreased by 3% in lactating females (D). There was a dose-dependent effect of Moringa oleifera aqueous seed extract on body weight gain in pregnant females (E) except at the 2nd week where animals of the control group and those submitted to heat stress alone showed a greater body weight gain. However, in lactating females (F), the animals of the control group showed the highest body weight gain (8%) compared to those submitted to heat stress, except at the 1st week of lactation where animals treated with 200 mg/kg b.w. of Moringa oleifera aqueous seed extract had the highest body weight gain. In these animals, body weight gain decreased weekly until weaning while animals solely submitted to heat stress showed loss in body weight from the 2nd week of treatment.

The results of Table 2 showed that voluntary mating was the most observed type of mating in female rabbits even though hand mating increased with the heat stress. Generally, most of animals of the control group required one service to achieve pregnancy (71.4%), while 42.9% of animals submitted to heat stress and co-treated with 100 and 200 mg/kg b.w. Moringa oleifera aqueous seed extract, respectively, required two services to achieve pregnancy and most of animals submitted to heat stress alone required two services to become pregnant (71.4%). The self fur removal was intense in most animals of control group (71.4%), lightly intense in animals co-treated with 100 mg/kg b.w. (57.1%) and 200 mg/kg b.w. Moringa oleifera aqueous seed extract (85.7%), while most of animals of the group solely submitted to heat stress did not remove their fur (57.1%). A significant decrease in the litter size was observed in animals submitted to heat stress compared to those of control group. However, this parameter increased in a dose-dependent manner following administration of Moringa oleifera aqueous seed extract. There was no group significant difference in the gestation length.

The number of kids was significantly higher in female rabbits from the control group from birth to weaning (35 days) compared to those submitted to heat stress. However, in animals submitted to heat stress and co-treated with Moringa oleifera aqueous seed extract, this parameter increased in a dose-dependent manner without reaching the values of the control group (Table 3).

The results of Table 4 showed a significant increase in litter weight of the control group from birth to weaning (35 days) compared to groups submitted to heat stress. However, when co-treated with Moringa oleifera aqueous seed extracts, this parameter increased in a dose-dependent manner but without achieving the values of the control group. The kit body weight at weaning (35 days) and the daily kit weight gain indicated a significant increase in the control group compared to groups submitted to heat stress. Nevertheless, in heat-stressed animals, these parameters were increased in female rabbits co-treated with 100 and 200 mg/kg b.w. Moringa oleifera aqueous seed extract, respectively, compared to those submitted to heat stress alone.

The results indicate a significant increase in milk yield in female of the control group from birth to weaning (35 days) compared to groups submitted to heat stress. However, in animals submitted to heat stress and co-treated with Moringa oleifera aqueous seed extract, this parameter increased in a dose-dependent manner without reaching the values of the control group. The daily kit milk intake of the 1st and 2nd weeks was high in animals of the control group and those receiving 100 and 200 mg/kg b.w. Moringa oleifera aqueous seed extract, respectively, compared to those only submitted to heat stress; the opposite trend was observed at the fourth and 5th weeks, while there was not any significant difference at the 3rd week. Except the 3rd week where there was no significant difference, the milk efficiency significantly increased in females of the control group and those co-treated with 100 and 200 mg/kg b.w. Moringa oleifera aqueous seed extract, respectively, during the first, second, fourth, and fifth weeks compared to the group submitted to the heat stress alone (Table 5).

The results of Figure 2 indicated that serum levels of estradiol and FSH significantly decreased in the heat-stressed animals compared to the control group. These parameters increased in heat-stressed animals, co-treated with 100 and 200 mg/kg b.w. Moringa oleifera aqueous seed extract, respectively, compared to those submitted to heat stress alone. The opposite trend was observed in serum progesterone and cortisol levels. On the contrary, LH content was decreased (P < 0.05) in animals submitted to heat stress alone compared to those of the control group and those receiving 100 and 200 mg/kg b.w. Moringa oleifera aqueous seed extract, respectively. There was no significant difference in serum prolactin content.

The ovary of animals (Figure 3) from control group (A) was characterized by the presence of a dominant growing follicles with oocyte, forming antral cavity, and surrounded by atretic follicles. However, the ovary of animals submitted to heat stress and co-treated with 200 mg/kg b.w. of Moringa Oleifera aqueous extract (C) showed multifocal areas of follicular degeneration characterized with oocyte lysis in graafian follicle, degeneration of granulosa cells and theca cells, and disorganization of corona radiate and cumulus oophorus cells. Animals submitted to heat stress and receiving 100 mg/kg b.w. of Moringa Oleifera aqueous extract (B) showed a marked degeneration of graafian ovarian follicle having eosinophil amorphous material (oocyte lysis), severe degeneration of corona radiata cells, granulosa cells, and cumulus oophorus. The tissues from the animals exposed only to heat stress (D) showed focal areas of proliferation of fibroblast in the interstitial tissues, in addition to severe follicular degeneration characterized by oocyte lysis and accompanying enhanced general follicular cell degeneration.