Black Maca (Lepidium meyenii Walp.) Hydroalcoholic Extract as an Ameliorating Agent against Heat Stress Conditions of V-Line Rabbit Does

Abstract

Rabbits are sensitive to heat stress (HS) in hot regions due to difficulty in eliminating excess body heat. We evaluated the ameliorating role of black maca hydroalcoholic extract (BMHE) against HS conditions on the productive and reproductive performance of V-line rabbit does. Rabbits were divided into four equal groups (T₁–T₄), each containing three replicates. T₁ received commercial basal diet (BD) only, whereas T₂–T₄ received BD and 200, 400, and 600 mg BMHE kg⁻¹ body weight (BW) of doe day⁻¹, respectively, administered orally for 1 week before the mating process each month from May to August. HS significantly decreased the BW of rabbits after the weaning period, as well as litter size, and litter weights measured 7, 14, 21, and 28 days after the postnatal period. HS conditions also significantly decreased BW at slaughter as well as all carcass quality parameters. HS led to significantly impaired physiological responses, oxidative status, and reproductive efficiency in exposed rabbits. Orally administered 400 mg BMHE kg⁻¹ BW of doe alleviated all these drastic effects in HS rabbits among all treatments. Thus, oral treatment of 400 mg BMHE kg⁻¹ BW (T₃) is a promising ameliorating agent against HS conditions in V-line rabbit does, especially in tropical or subtropical regions.

1. Introduction

Rabbit production is one of the fast-growing projects in Egypt, as rabbits are quiet and highly prolific animals with a short gestation period between 31 and 33 days, fast growth, efficient feed utilization, and high fecundity [1]. They also provide high-quality protein and healthy meat, which is in high demand [2]. Egypt is one of the 10 leading rabbit meat producing countries; it produces 56 metric tons and contributes 3.8% to the global production of rabbit meat [3]. Thus, successful rabbit projects have been recorded in developing countries such as Egypt, which contribute to food security and income [4]. However, rabbits are highly sensitive to extreme environmental conditions, particularly temperature. Thermoregulation in rabbits is somewhat poor, as they have few functional sweat glands [5]. The optimal temperature range of rabbits is 15–25 °C, and the optimal humidity is 55–65%. Heat stress (HS) occurs when the ambient temperature is >30 °C. When the temperature is >35 °C, rabbits cannot regulate their body temperature, resulting in heat failure and heat-induced physiological stress [6]. Recently, a serious hazard to livestock production, including rabbits, has occurred due to global climate change, especially the expected increase in surface temperature [7].

In tropical and subtropical regions such as Egypt, HS is intensified with high relative humidity (RH), which is normally >85% during the day and can reach 100% during hot months [5]. HS has multiple unfavorable effects on rabbit health and production performance. It causes a 20–25% reduction in daily weight gain, an 8–15% decrease in feed conversion ratio, and a 9–12% increase in mortality rate. Moreover, the reproductive performance decreases by 6–10% and negatively influences meat quality and carcass traits [8]. Thus, HS causes a great challenge for the rabbit industry [5]. In addition, HS conditions caused drastic changes in the biological functions of exposed rabbits, including depression of immune responses, enzymatic activities, hormonal secretions, and blood metabolites, consequently impairing productive and reproductive efficiency [9]. Furthermore, HS markedly increases reactive oxygen species and free radicals (FR), reduces cellular antioxidants [10], increases corticosteroid levels, and decreases luteinizing hormone (LH) and follicle-stimulating hormone secretion, affecting ovary development and the rate of ovulation [11], and having adverse effects on DNA, proteins, and lipids [12]. Moreover, Villalobos et al. [13] reported that HS conditions prevent rearing rabbits in high density up to 18 rabbits m⁻², leading to a severe decline in rabbit production.

Amelioration of the drastic effects of HS on rabbits has been attempted using different procedures, including offering cool drinking water [14], using a short lighting regime [15], and providing vitamins in the diet [16] or drinking water [17]. Many medicinal plants can be used as promising growth promoters for improving the productive and reproductive performances of rabbits [18], especially under HS conditions, where herbs contain phytoestrogens as plant chemicals similar to sex hormones [19]. Additionally, medicinal plants have high antioxidant activity, and antioxidants play important roles in protecting animals against HS conditions. Thus, some medicinal plants have been used as protector agents against HS situations in rabbits, such as curcumin [20], a combination of royal jelly (RJ) and green tea (GT) [21], Emblica officinalis [22], Moringa oleifera extract [23], M. oleifera leaf powder [24], or ginger root powder [25].

Maca (Lepidium meyenii) is traditionally traded as capsules or powder worldwide due to its potential medicinal and nutritional activities [26]. These include its capacity to improve female [27] and male fertility and sexual performance [28], antioxidation [29], immunomodulatory activities [30], and cell protection [31]. Most certainly, these biological and pharmacological activities are mainly related to the chemical components of L. meyenii [32]. The main bioactive compounds in L. meyenii are categorized into six classes: glucosinolates and isothiocyanates, thiohydantoins, macaene and macamides, alkaloids, polysaccharides, and fatty acids [33]. There is a lack of sufficient research regarding the alleviative effects of the maca plant or its extracts on highly HS-sensitive farm animals such as rabbits, and especially females. Therefore, this study was designed to evaluate the ameliorative effects of black maca (L. meyenii) hydroalcoholic extract (BMHE) on the productive and reproductive performance of V-line rabbit does reared under HS conditions.

2. Materials and Methods

This study was conducted from May to August 2021 at the rabbit farm of the El-Serw Animal Production Research Station, Animal Production Research Institute, Agriculture Research Center, Ministry of Agriculture, Egypt.

2.1. Experimental Design

Thirty-six V-line rabbit does 11–12 months of age and weighing 2.74 kg were randomly divided into four equal groups. All experimental rabbits were healthy and clinically free from external and internal parasites. They were housed under the same environmental and managerial conditions throughout the experimental period (from May to September). Each group was represented in three replicates, where three does were individually housed in each replicate. The first group (T₁) was fed a commercial pelleted basal diet (BD) only, whereas the second (T₂), third (T₃), and fourth (T₄) groups were fed BD and orally administered with 200, 400, and 600 mg BMHE kg⁻¹ body weight (BW) of doe day⁻¹, respectively, for 1 week before mating in each month from May to August (which is the hot summer season in Egypt). All does were individually housed in wire galvanized battery cages (50 × 50 × 40 cm, length × width × height) in an open-side house under the same managerial and hygienic conditions. Cages were cleaned and disinfected regularly. Light in their houses was allowed 12–14 h daily during all experimental periods. Urine and feces dropped from the cages on the floor were cleaned daily in the morning. Each cage was provided with fresh water, and the experimental pelleted BD was offered ad libitum all over the experimental period.

2.2. Experimental Diet

All experimental rabbits were fed a commercial pelleted BD. The pelleted BD was formulated to meet all nutrient requirements of rabbit does as recommended by the NRC [34], was a nearly isonitrogenous and isocaloric-based diet of crude protein (CP) and digestible energy (DE), and was fortified with adequate vitamins and minerals. The formula and chemical analysis of the pelleted BD are shown in

Table 1. Composition and calculated analysis of the experimental basal diet.

Item	%
Ingredient
Barley grain	24.60
Alfalfa hay	31.00
Soybean meal	13.25
Wheat bran	28.00
Di-calcium phosphate	1.60
Limestone	0.95
Sodium chloride	0.30
Mineral-vitamin premix 1	0.30
Total	100
Chemical analysis 2 (as % on dry matter basis)
CP (%)	17.08
CF (%)	12.55
Ether extract (%)	2.20
DE (kcal/kg)	2416
Metabolizable energy (kcal/kg) 3	2219
Calcium (%)	1.20
Total phosphorus (%)	0.761
Lysine (%)	0.84
Methionine (%)	0.23

¹ One kilogram of minerals–vitamins premix provided as: Vitamin A, 150,000 IU; Vitamin E, 100 mg; Vitamin K3, 21 mg; Vitamin B1, 10 mg; Vitamin B2, 40 mg; Vitamin B6, 15 mg; Pantothenic acid, 100 mg; Vitamin B12, 0.1 mg; Niacin, 200 mg; Folic acid, 10 mg; Biotin, 0.5 mg; Choline chloride, 5000 mg; Fe, 0.3 mg; Mn, 600 mg; Cu, 50 mg; Co, 2 mg; Se, 1 mg; and Zn, 450 mg. ² Chemical analysis according to feed composition tables for rabbits’ feedstuffs used by De Blas and Wiseman [35]. ³ ME (kcal/kg diet) estimated as 0.95 DE according to Santoma, et al. [36].

. The tested BMHE was purchased from AbChem (Mansoura, Egypt) for pharmaceutical raw materials. It consists of 10.2% CP, 59.0% carbohydrate, 2.20% crude fat, and 8.5% crude fiber (CF).

2.3. Climatic Conditions

This study was carried out from May to August 2020, when the highest temperature-humidity index (THI) is observed during the hot summer season in Egypt. The interior ambient temperature (°C) and RH (%) were recorded daily each month during all experimental periods on the rabbits’ farm at 9 A.M. and 1 P.M. using a thermohygrometer.

THI was calculated according to the same procedure for rabbits using the following equation according to Maria et al. [14]:

$$\mathrm{THI}= \mathrm{t}-\left[\left(0.31-0.31 \left(\frac{\mathrm{RH}}{100}\right)\right)\times \left(\mathrm{t} -14.4\right)\right]$$

where t is the dry bulb temperature (°C). THI values were classified as <27.8 (absence of HS), 27.8–28.9 (moderate HS), 29.0–30.0 (severe HS), and >30.0 (very severe HS).

2.4. Tested Measurements

2.4.1. V-Line Rabbit Does and Their Litter Traits

In the last week of each month from June to August of the growing period, all V-line rabbit does in each group (n = 9) were individually weighed and orally administered with different levels of BMHE kg⁻¹ BW of doe day⁻¹ for 1 week before mating in each month during the experimental period. The mating process was conducted with the black maca untreated rabbit does reared under the same conditions on the same animal farm. After 28 days of mating each month, the fertilized rabbit does were born, where the litter weight was measured 7, 14, 21, and 28 days after birth, and the mortality rate of the litter (0–28 days after birth) was also calculated. After the weaning period, rabbit does were individually weighed.

2.4.2. Carcass Quality Traits

At the end of the growing period, six growing rabbits from each group (n = 6) were fasted for 12 h and weighed, the slaughtered animals were deskinned and dressed out, and the hot carcass, including the head, was weighed. The edible parts (head, liver, heart, kidneys, and fore parts) were separately weighed. The dressing percentage was calculated using the following equation:

$$\mathrm{Dressing} (\%)=100\times \left(\frac{\mathrm{Carcass} \mathrm{weight} + \mathrm{head}}{\mathrm{Live} \mathrm{body} \mathrm{weight}}\right)$$

2.4.3. Hematological and Serum Biochemical Analysis

At the end of the experiment, the same six rabbits used in the carcass quality traits (n = 6) in each treatment were also taken to collect blood samples. Fresh blood samples were collected from the rabbit’s ear vein in heparinized test tubes to determine the hematological parameters. Blood samples were also collected without anticoagulant and kept at room temperature. The tubes were centrifuged at 3500 rpm for 20 min to separate the clear serum. The serum samples were kept in a deep freezer (−20 °C) until serum biochemical analysis was carried out.

Hematological parameters such as hemoglobin (Hb) were determined using commercial kits (Diamond Diagnostic, Cairo, Egypt). Total red blood cells (RBCs), total white blood cells (WBCs), and the differential of WBCs were counted according to Dacie and Lewis [37] on an Ao Bright-Line Hämocytometer model (Neubauerimroved, Precicolor HBG, Giessen, Germany) and the packed cell volume (PCV %) was measured. Hematological indices, such as mean cell volume (MCV), mean cell Hb (MCH), and MCH concentration (MCHC), were calculated according to Dacie and Lewis [37]. Serum biochemical parameters, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, albumin, globulin, total bilirubin, and uric acid, were determined using Diamond Diagnostic kits according to Reitman and Frankel [38]. Serum total cholesterol (TC) was measured according to Tietz and Berger [39]. Triglycerides were measured according to McGowan et al. [40]. High-density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (vLDL) were measured according to Warnick and Wood [41]. Serum total antioxidant capacity (TAC) was measured according to Koracevic et al. [42]. Glucose was measured according to Henry [43]. Serum cortisol, estrogen (E2), and progesterone (P4) were measured using the commercial ELISA test kits catalog number M-1850 (Alpha Diagnostic International, San Antonio, TX, USA), BC-1111, and BC-1113 (BioCheck, Inc., San Francisco, CA, USA), respectively, according to Tietz [44].

2.5. Statistical Analysis

All data were presented as the mean ± standard error of the mean and statistically analyzed using SAS^® version 9.1.3 for Windows [45] according to the following model:

$${\mathrm{Y}}_{\mathrm{ik}}=\mathsf{\mu } +{ \mathrm{T}}_{\mathrm{i}}+{ \mathrm{e}}_{\mathrm{ik}}$$

where μ = the overall mean, T_i = fixed effect of treatments [1 = 0 maca extract (control), 2 = 200 mg maca extract, 3 = 400 mg maca extract, and 4 = 600 mg maca extract], and e_ik = residual error. The significant differences among means were compared using Duncan’s new multiple range test [46]. Treatments were considered significantly different if p < 0.05.

3. Results

3.1. THI

In this study, the climatic conditions during all experimental periods are shown in

Table 2. Monthly average air temperature, humidity, and temperature–humidity index (THI) in the farm area during all experimental periods.

Month	Air Temperature (°C)	Humidity (%)	THI	Status of HS (According to [14])
May	28	61.86	26.39	Absence
June	31	74.63	29.69	Severe
July	33	77.51	31.70	Very severe
August	32	79.33	30.87	Very severe

, where the calculated THI ranged between 26.39 and 31.70. The THI values indicated that experimental rabbits were exposed to severe and very severe HS during the experimental period, especially from June to August.

3.2. Rabbit Does and Their Litter Traits

The effects of different levels of maca plant extract on V-line rabbit does reared under HS conditions and their litter traits at different intervals experimental periods (June-August) are illustrated in

Table 3. Effects of different levels of black maca hydroalcoholic extract (BMHE) on V-line rabbit does and their litter traits in June.

Parameters	T1 (Control)	T2	T3	T4
Female weight at mating (g)	2725.35 ± 50.99	2827.77 ± 47.22	2923.22 ± 77.50	2916.66 ± 52.70
Litter size	1.77 b ± 0.46	1.66 b ± 0.55	4.44 a ± 0.37	1.66 b ± 0.55
Litter mortality rate (%; 0–28 days)	74.07 a ± 14.46	66.67 a ± 16.66	8.15 b ± 5.42	66.67 a ± 16.66
Litter weight (g)
At 7 days	77.22 b ± 19.42	40.00 b ± 20.00	161.11 a ± 0.73	53.33 b ± 21.08
At 14 days	135.16 b ± 33.82	58.88 b ± 29.74	275.00 a ± 15.72	66.66 b ± 33.33
At 21 days	84.44 b ± 42.23	67.77 b ± 33.90	388.88 a ± 7.34	100.00 b ± 50.00
At 28 days	144.44 b ± 73.33	183.33 b ± 92.04	562.22 a ± 24.93	133.33 b ± 66.66
Female weight after weaning (g)	2790.55 b ± 24.60	2961.11 a ± 50.53	3010.66 a ± 36.78	2990.00 a ± 75.93

Mean in the same row within each item having different superscripts are significantly different (p ≤ 0.05). Each value represents as the mean ± standard error (n = 9). T₁ = Rabbits fed the basal diet (BD); T₂ = Rabbits fed BD and orally administrated with 200 mg BMHE kg⁻¹ BW of doe day⁻¹; T₃ = Rabbits fed BD and orally administrated with 400 mg BMHE kg⁻¹ BW of doe day⁻¹; T₄ = Rabbits fed BD and orally administrated with 600 mg BMHE kg⁻¹ BW of doe day⁻¹.

,

Table 4. Effects of different levels of BMHE on V-line rabbit does and their litter traits in July.

Parameters	T1 (Control)	T2	T3	T4
Female weight at mating (g)	2990.00 ± 75.93	2755.55 ± 73.80	2951.77 ± 47.75	3030.00 ± 42.26
Litter size	1.11 b ± 0.51	1.22 b ± 0.43	5.11 a ± 0.26	1.66 b ± 0.60
Litter mortality rate (%; 0–28 days)	63.88 b ± 16.20	44.44 bc ± 17.57	10.92 c ± 5.60	100.00 a ± 0.00
Litter weight (g)
At 7 days	55.00 b ± 21.76	90.00 ab ± 28.52	148.33 a ± 6.77	53.33 b ± 21.08
At 14 days	78.88 b ± 31.34	126.11 ab ± 40.90	207.77 a ± 2.22	80.00 b ± 31.63
At 21 days	117.22 b ± 46.55	164.83 b ± 53.28	415.55 a ± 14.63	64.44 b ± 42.65
At 28 days	160.77 b ± 64.27	200.83 b ± 66.10	504.44 a ± 10.15	0.00 c ± 0.00
Female weight after weaning (g)	2822.22 b ± 65.14	2866.66 ab ± 39.96	2961.66 a ± 34.11	2839.44 ab ± 27.13

Mean in the same row within each item having different superscripts are significantly different (p ≤ 0.05). Each value represents as the mean ± standard error (n = 9). T₁ = Rabbits fed the basal diet (BD); T₂ = Rabbits fed BD and orally administrated with 200 mg BMHE/kg BW of doe day⁻¹; T₃ = Rabbits fed BD and orally administrated with 400 mg BMHE kg⁻¹ BW of doe day⁻¹; T₄ = Rabbits fed BD and orally administrated with 600 mg BMHE kg⁻¹ BW of doe day⁻¹.

and

Table 5. Effects of different levels of BMHE on V-line rabbit does and their litter traits in August.

Parameters	T1 (Control)	T2	T3	T4
Female weight at mating (g)	2855.55 ± 59.18	2866.66 ± 39.96	2845.00 ± 33.80	2845.00 ± 36.57
Litter size	2.00 b ± 0.64	1.33 b ± 0.55	4.66 a ± 0.28	1.55 b ± 0.47
Litter mortality rate (%; 0–28 days)	90.74 a ± 6.28	55.55 ab ± 17.57	38.51 b ± 8.57	33.33 b ± 16.67
Litter weight (g)
At 7 days	68.33 b ± 21.63	68.88 b ± 27.30	165.22 a ± 3.98	84.44 b ± 21.22
At 14 days	111.11 b ± 35.13	75.55 b ± 37.93	342.11 a ± 16.84	135.55 b ± 34.92
At 21 days	67.77 c ± 44.83	98.88 bc ± 49.45	408.88 a ± 25.35	206.66 b ± 51.93
At 28 days	83.33 c ± 55.27	127.77 bc ± 64.06	508.88 a ± 68.24	280.00 b ± 70.29
Female weight after weaning (g)	2855.55 ± 59.18	2866.66 ± 39.96	2872.22 ± 33.44	2845.00 ± 36.57

Mean in the same row within each item having different superscripts are significantly different (p ≤ 0.05). Each value represents as the mean ± standard error (n = 9). T₁ = Rabbits fed the basal diet (BD); T₂ = Rabbits fed BD and orally administrated with 200 mg BMHE kg⁻¹ BW of doe day⁻¹; T₃ = Rabbits fed BD and orally administrated with 400 mg BMHE kg⁻¹ BW of doe day⁻¹; T₄ = Rabbits fed BD and orally administrated with 600 mg BMHE kg⁻¹ BW of doe day⁻¹.

, respectively. In June and July, no significant (p ≥ 0.05) differences were detected in the weight (g) of female rabbit does at mating among all treatments (Table 3 and Table 4). Meanwhile, those reared under HS conditions and orally administered with different levels of BMHE kg⁻¹ BW of doe day⁻¹ have heavier (p ≤ 0.05) weights (g) after the weaning period than the control group (T₁). Regarding litter traits, rabbits reared under HS conditions in June and July and orally administered with 400 mg BMHE kg⁻¹ BW of doe day⁻¹ (T₃) achieved the lowest litter mortality rate (%; 0–28 days), largest litter size, and heavier litter weights measured 7, 14, 21, and 28 days after the postnatal period among all treatments (Table 3 and Table 4; p ≤ 0.05).

In August, weight at mating and after weaning of V-line rabbit does reared in very severe HS conditions were not significantly different among all treatments (Table 5). Regarding litter traits, HS rabbits orally administered with 400 mg BMHE kg⁻¹ BW of doe day⁻¹ (T₃) had the highest (p ≤ 0.05) litter size and heavier litter weights measured 7, 14, 21, and 28 days after the postnatal period among all treatments (Table 5). However, rabbits reared in very severe HS conditions and orally administered with 600 and 400 mg BMHE kg⁻¹ BW of doe day⁻¹ (T₄ and T₃), respectively, had the lowest (p ≤ 0.05) litter mortality rate (%; 0–28 days) than those fed BD only as a control treatment (T₁).

3.3. Carcass Quality Traits

Table 6. Effects of different levels of BMHE on carcass quality traits of V-line rabbit does.

Parameters	T1 (Control)	T2	T3	T4
BW at slaughter (g)	2654.00 bc ± 56.31	2850.00 ab ± 56.34	2933.33 a ± 66.66	2633.33 c ± 72.64
Head (g)	130.66 b ± 0.66	135.66 ab ± 2.33	143.66 a ± 1.85	136.66 ab ± 6.00
Thigh (g)	460.00 b ± 5.77	498.33 ab ± 1.66	541.66 a ± 10.92	516.66 a ± 23.15
Front parts (g)	369.00 ± 0.99	375.00 ± 2.88	383.33 ± 16.66	386.66 ± 13.33
Trunk (g)	444.00 c ± 1.20	491.66 b ± 6.00	557.66 a ± 3.71	486.66 b ± 24.03
Spleen (g)	1.40 c ± 0.05	2.10 ab ± 0.05	1.95 b ± 0.02	2.16 a ± 0.08
Kidneys (g)	15.83 ± 0.18	17.60 ± 0.19	16.74 ± 0.74	15.26 ± 2.21
Liver (g)	64.93 c ± 1.35	67.40 bc ± 0.80	76.80 a ± 1.00	70.81 b ± 1.43
Heart (g)	5.45 c ± 0.09	6.94 b ± 0.17	8.95 a ± 0.29	6.91 b ± 0.02
† Dressing (g)	1403.66 b ± 7.79	1500.66 b ± 6.35	1626.33 a ± 30.71	1526.66 ab ± 37.90

Mean in the same row within each item having different superscripts are significantly different (p ≤ 0.05). Each value represents as the mean ± standard error (n = 6). T₁ = Rabbits fed the basal diet (BD); T₂ = Rabbits fed BD and orally administrated with 200 mg BMHE kg⁻¹ BW of doe day⁻¹; T₃ = Rabbits fed BD and orally administrated with 400 mg BMHE kg⁻¹ BW of doe day⁻¹; T₄ = Rabbits fed BD and orally administrated with 600 mg BMHE kg⁻¹ BW of doe day⁻¹. ^† Dressing (g) = (Head + Thigh + Front parts + Trunk).

presents the effects of maca extract on carcass quality traits of V-line rabbit does reared under HS conditions. HS rabbits fed BD and orally administered with 400 mg BMHE kg⁻¹ BW of doe day⁻¹ (T₃) significantly (p ≤ 0.05) increased BW at slaughter and all carcass quality parameters among other treatments, except for the front parts and kidneys, which were not significantly affected among all treatments.

3.4. Hematological Parameters

Table 7. Effects of different levels of BMHE on hematological parameters of V-line rabbit does.

Parameters	T1 (Control)	T2	T3	T4
Hemoglobin (Hb, g dL−1)	8.40 d ± 0.05	9.10 c ± 0.08	12.50 a ± 0.20	9.80 b ± 0.11
Red blood cells (RBCs, ×1012 L−1)	4.32 c ± 0.01	4.61 b ± 0.05	5.38 a ± 0.01	5.36 a ± 0.01
Packed cell volume (PCV, %)	27.23 c ± 0.18	30.53 b ± 0.63	33.96 a ± 0.92	32.10 b ± 0.05
Mean corpuscular volume (MCV, µm3)	65.02 b ± 0.56	68.00 a ± 0.47	60.28 c ± 0.03	59.42 c ± 0.32
Mean corpuscular hemoglobin (MCH, pg cell−1)	19.18 a ± 0.09	19.49 a ± 0.26	18.21 b ± 0.19	18.04 b ± 0.01
Mean corpuscular hemoglobin concentration (MCHC, %)	29.71 b ± 0.32	29.52 b ± 0.28	30.88 a ± 0.01	30.17 ab ± 0.04
Blood platelets (×109 L−1)	133.00 b ± 1.45	140.00 b ± 2.88	244.66 a ± 2.33	240.00 a ± 0.57
White blood cells (WBCs, ×109 L−1)	3.80 d ± 0.05	5.40 c ± 0.11	11.20 a ± 0.11	7.70 b ± 0.05
Neutrophils (%)	38.00 d ± 0.57	42.36 c ± 0.18	50.66 a ± 0.33	48.00 b ± 0.57
Lymphocytes (%)	38.66 c ± 0.33	40.66 c ± 0.66	50.33 a ± 0.33	45.76 b ± 0.22
Monocytes (%)	5.00 c ± 0.00	6.00 b ± 0.00	8.00 a ± 0.00	7.00 a ± 0.33
Eosinophils (%)	3.00 b ± 0.00	3.00 b ± 0.00	4.00 a ± 0.00	3.66 a ± 0.33
Basophils (%)	0.00 b ± 0.00	0.00 b ± 0.00	1.33 a ± 0.33	0.00 b ± 0.00

Mean in the same row within each item having different superscripts are significantly different (p ≤ 0.05). Each value represents as the mean ± standard error (n = 6) T₁ = Rabbits fed the basal diet (BD); T₂ = Rabbits fed BD and orally administrated with 200 mg BMHEkg⁻¹ BW of doe day⁻¹; T₃ = Rabbits fed BD and orally administrated with 400 mg BMHE kg⁻¹ BW of doe day⁻¹; T₄ = Rabbits fed BD and orally administrated with 600 mg BMHE kg⁻¹ BW of doe day⁻¹.

shows the hematological parameters of HS rabbits orally treated with different levels of BMHE (T₂–T₄) and those fed BD only as a control treatment (T₁). Rabbits orally treated with 400 and 600 mg BMHE kg⁻¹ BW of doe day⁻¹ (T₃ and T₄, respectively) had significantly (p ≤ 0.05) increased RBCs, MCHC, platelets, monocytes, and eosinophils than other treatments (T₁ and T₂). However, those orally treated with 400 mg BMHE kg⁻¹ BW of doe day⁻¹ (T₃) had significantly (p ≤ 0.05) increased Hb, PCV, WBCs, neutrophils, lymphocytes, and basophils compared to other treatments. Meanwhile, rabbits orally treated with 200 mg BMHE kg⁻¹ BW of doe day⁻¹ (T₂) had significantly increased MCV compared to treatments, but MCH significantly increased in control (T₁) or T₂ treatment.

3.5. Serum Biochemical Parameters

3.5.1. Liver and Kidney Function Traits

Rabbits orally treated with 400 mg BMHE kg⁻¹ BW of doe day⁻¹ (T₃) and reared in HS conditions had significantly (p ≤ 0.05) decreased serum AST and ALT (Figure 1A) among all treatments. However, HS rabbits in T₃ and T₁ had significantly (p ≤ 0.05) increased serum total protein and albumin than those in T₂, but no significant (p ≥ 0.05) differences in serum total protein and albumin were found between rabbits in T₂ and T₄ (Figure 1B). No significant (p ≥ 0.05) differences in serum globulin among all treatments were noticed (Figure 1B). Regarding serum total bilirubin, rabbits fed BD as a control treatment (T₁) reared under HS conditions had significantly increased total bilirubin among all treatments (Figure 1C; p ≤ 0.05). Meanwhile, rabbits reared in HS conditions in T₁ and T₂ had significantly increased serum uric acid than those in T₃ and T₄ (Figure 1D; p ≤ 0.05).

3.5.2. Lipid Profile Parameters

Figure 2A and B illustrates the effects of the different levels of orally administered BMHE to HS rabbits on serum lipid profile indicators. HS rabbits orally treated with 400 and 600 mg BMHE kg⁻¹ BW of doe day⁻¹ (T₃ and T₄, respectively) had significantly (p ≤ 0.05) decreased serum TC than those in T₁ and T₂ (Figure 2A). In the same trend, serum triglycerides (Figure 2A), LDL, and vLDL (Figure 2B) of HS rabbits in T₃ also significantly (p ≤ 0.05) decreased among all treatments. In a converse trend, rabbits in the same treatment T₃ had significantly (p ≤ 0.05) increased serum HDL among all treatments.

3.5.3. TAC, Glucose, Cortisol, and Sex Hormones Parameters

Serum TAC significantly (p ≤ 0.05) increased in rabbits reared in HS conditions and orally treated with 400 and 600 mg BMHE kg⁻¹ BW of doe day⁻¹ (T₃ and T₄, respectively) than those in T₁ and T₂ (Figure 3A). HS rabbits fed BD only as a control treatment (T₁) had significantly (p ≤ 0.05) increased serum cortisol hormone (Figure 3C) among all treatments. HS rabbits in T₃ had the lowest (p ≤ 0.05) serum glucose (Figure 3B) among all treatments. In an inverse trend, rabbits reared in HS conditions in the same treatment (T₃) had the highest (p ≤ 0.05) serum E2 (Figure 3D) and P4 (Figure 3E) hormones among all treatments.

4. Discussion

In this study, the calculated THI ranged between 29.69 and 31.70 from June to August, indicating that experimental rabbits were exposed to severe and very severe HS during the experimental period, according to the classification of THI by [14]. Moreover, Marai et al. [47] suggested that the optimal THI for rabbit husbandry is 27.8. The thermal stress on rabbits can be estimated by THI, which combines the effects of ambient air temperature and RH, and is used to assess the severity of HS. Thus, Marai et al. [48] stated that THI is the most used index stress to evaluate the effects of climatic conditions and assess the effects of HS on livestock production.

Litter size at birth and weaning is an important economic trait which determines the number of kits for future breeding programs and lifetime production [49]. In this study, V-line rabbit does and their litter traits were adversely affected by exposure to severe and very severe HS conditions from June to August. Similarly, Marco Jiménez et al. [50] reported that litter size, litter weight, and kit birth weight in rabbits were reduced in those whose dams were exposed to HS conditions during gestation compared to unstressed does. In a recent study, Mady et al. [51] reported differences in birth and weaning weights in rabbits between the winter and summer seasons. Rabbits had a heavier weight at birth (53.38 g) and weaning (374.44 g) during winter than in summer (37.56 and 304.78 g, respectively). The present study showed that the oral administration of BMHE at level 400 mg kg⁻¹ BW of doe day⁻¹ significantly (p ≤ 0.05) increased the rabbit doe’s weight after the weaning period, as well as increased the litter size and litter weight measured at 7, 14, 21, and 28 days after the birth of heat stressed rabbits at June, July and August, compared to other treatments. This effect seems to be due to an effect favoring the survival of embryos. Thus, it can be noted that oral administration of BMHE at level 400 mg kg⁻¹ BW of doe day⁻¹ seriously alleviated the drastic effects of HS on rabbit does and their litters. The positive effects of maca were similarly observed in mice [27], rats [52], and murine [53]. These positive effects may be due to maca’s sterol bioactive components, such as campesterol (27.3%), ergosterol (13.6%), brassicasterol (9.1%), ^Δ7,22-ergostadienol (4.5%), and sitosterol (45.5%), having phytoestrogen activity [54]. Recently, El-Sheikh et al. [55] reported that oral treatment of maca capsules in rabbit does insignificantly affected the number of matings per conception, conception rate, and gestation length, and had no significant effects on the litter weight of maca-treated rabbit does at 7, 21, and 28 days; however, those at 14 days were insignificantly lower than the control group [49]. The disparities between this study and others may be due to the differences in the experimental animals’ age, maca extract and its levels, exposure time, and experimental management. The litter mortality rate significantly reduced throughout the experimental periods in treated rabbits with BMHE at level 400 mg kg⁻¹ BW of doe day⁻¹. This may be due to the improvement of some hematological and biochemical parameters in this group. The 100% mortality with the supplement at 600 mg kg⁻¹ BW of doe day⁻¹ (T₄) in July resulted because the rabbit does in T₄ showed a sudden nervous state as a result of exposure to heat stress, thus they killed the whole litter that reared with them in the same cage.

Rabbits are susceptible to HS, which is associated with reductions in growth performance, feed intake, and feed efficiency parameters [56]. Thus, this study detected negative effects on growth performance and carcass quality parameters in HS-exposed rabbits. In the same line, a significant reduction in slaughter, carcass, and organ weight was noticed in rabbits subjected to HS [57]. Thus, many studies suggested that the decrease in growth performance of experimental animals caused by thermal stress may be a consequence of damage to the intestinal mucosa and the increase in the hydrolysis of muscle proteins caused by temperature-induced FR [13,58]. Moreover, Obled et al. [59] reported that stress conditions could increase the demand for some amino acids due to the synthesis of specific proteins, selective catabolism, or use in the synthesis of specific molecules. In this study, the improved growth and carcass quality traits of maca-treated rabbit does could be attributed to the properties of the tested maca extract, which acts as an antibacterial, antiprotozoal, antifungal, antioxidant, or immunostimulant agent [60].

The assessment of hematological parameters could be useful in determining the rabbits’ health status [61]. It is worth to be mentioned that all the hematological measurements of the experimental rabbits in the current study were within the normal range for healthy rabbits according to [62,63]. Similarly, these findings recorded great perturbation in hematological and serum biochemical parameters in New Zealand White (NZW) rabbits exposed to hot temperatures [64]. RBCs and PCV were lowered by an ambient temperature of 36 °C over 1 month in adult NZW rabbits [65], lower than the values obtained under the thermoneutral zone (18–21 °C) of rabbits. In general, animals exposed to various stresses have shown an increase in heterophils (H) and a decrease in lymphocytes (L), leading to an increase in the H/L ratio [66]. In this regard, Ismaiel [67] explained that the decrease in the H/L ratio in tropical conditions might be due to the requirements of extra amino acids, probably reflecting the synthesis of proteins or other specific compounds, such as hormones and heat shock proteins 70 that can ameliorate the negative effects of HS. Regarding the mitigation of negative effects of HS, the current findings indicated that rabbits orally treated with 400 and 600 mg BMHE kg⁻¹ BW of doe day⁻¹ (p ≤ 0.05) showed a significant increase in hematological parameters compared to other treatments. These positive effects of maca extract on the hematological parameters of heat stressed rabbits in the present study may justify the positive relationship between nutrients and the health biomarkers of farm animals, including rabbits. Furthermore, maca has some phytochemical components, such as polyphenolic, alkaloids, and flavonoids, which have positive impacts on different hematological measurements that could be attached to the cellular plasma membranes to protect the body cells from the produced FR, and/or activate the antioxidant enzymes [68]. In the same trend as this study, El-Ratel et al. [69] recently reported that dietary administration of curcumin or nano-curcumin significantly improved the hematological parameters of HS-exposed rabbits.

In this study, severe and very severe HS conditions led to harmful effects on all serum tested parameters of exposed rabbits, reflecting the dysfunctions of hepatic and renal organs, as well as impaired immune responses and antioxidant biomarkers in HS rabbits [70]. Similar to this study, Gabr et al. [21] stated that liver enzyme activities (AST and ALT) significantly increased in NZW weaned rabbits exposed to HS, suggesting some liver damage in mammals and birds, as cited by Faisal et al. [71]. Liang et al. [6] recently reported that blood biochemical parameters play critical roles in reflecting the metabolic changes and organ damage of rabbits under HS situations. Thus, total protein, glucose, and triglyceride concentrations in the blood decrease, whereas TC concentrations are markedly increased in rabbits exposed to HS [6]. Continuous exposure of rabbits to HS causes severe disruptions in homeostatic mechanisms, damages various organs [72], makes rabbits vulnerable to pathogens, and consequently brings serious losses to rabbit production [73]. In the present study, orally treated BMHE at level 400 mg kg⁻¹ BW of doe day⁻¹ significantly improved the tested serum biochemical parameters of HS-exposed rabbits. Reduced serum AST and ALT activities and increased total protein and albumin in orally treated BMHE groups reflect the beneficial effects of maca extract on liver function, alleviating the drastic effects of HS on exposed rabbits by increasing oxygen flow to the liver [74]. According to Gabr et al. [21], a combination of RJ and GT reduced the serum AST activity and increased the thyroid hormones of NZW weaned rabbits reared during a hot summer season in Egypt. According to [58], the high dose of dried Xinjiang maca powder (1200 mg kg⁻¹ BW) has harmful effects on the serum parameters of treated mice. The different results may be related to the different planting sources, types, dosages, and chemical composition.

HS conditions lead to the generation of FR, such as O₂− and HO, which can damage cell membranes by inducing lipid peroxidation of polyunsaturated fatty acids in the cell membrane [6]. In this study, exposure to HS and oral administration of maca extract in rabbits would have weakened the deleterious heat-induced oxidative stress, with significantly decreased serum TC, triglycerides, LDL, and vLDL, and significantly increased serum HDL. In this respect, Gabr et al. [21] reported that an oral combination of RJ and GT achieved the best findings of productive performance and physiological response parameters of HS NZW weaned rabbits. As in this study, Elnagar et al. [75] observed that RJ treatment significantly reduced serum total lipids, TC, triglycerides, creatinine, and uric acid concentrations in growing rabbits or male rabbits [76] treated with RJ at 50, 100 and 150 mg kg⁻¹ BW compared to HS control rabbits. Several authors indicated that the bioactive compound content in GT [77] and RJ [78] in their studies (or black maca in this study) such as polyphenols, could inhibit the key enzymes involved in the biosynthesis and absorption of lipids, triglycerides, and cholesterol as well as energy expenditure stimulation, fat oxidation, HDL concentrations, and lipid excretion in feces [32].

This study detected adverse effects on oxidative damage and stress-related biomarkers in HS-exposed rabbits. The current findings also indicated that oral administration of BMHE at level 400 mg kg⁻¹ BW led to enhance the profile of heat stressed rabbits, which indicates that BMHE can be used to reduce HS-induced oxidative damage and alleviate the drastic effects of HS on rabbits. This level of BMHE significantly increased serum TAC, while significantly decreasing both serum cortisol hormone and glucose in HS-exposed rabbits. These results indicated the useful application of BMHE as an viable protocol for rabbits reared in hot regions such as Egypt. These positive effects may be due to BMHE containing substantial amounts of antioxidant compounds, especially phenols, glucosinolates, alkamides, and polysaccharides [79]. In a recent study, Tafuri et al. [80] reported that specific bioactive metabolites in maca extract, such as glucosinolates and macamides, might be responsible for the antioxidant activity of maca. In this respect, black maca also reduced glucose levels, and consumption of this variety is associated with lower blood pressure and an improved health score [81]. In the same line, Choi et al. [82] reported that rats treated with a lipid-soluble extract of maca showed the highest levels of catalase in the liver and glutathione antioxidants in muscle and liver than in the control group. They also suggested that this effect depends on the effects of maca extracts on suppressing oxidative stress.

The endocrine system plays an important role in the animal’s response to stress, including HS [6]. When rabbits are exposed to HS, the hypophysis–pituitary–adrenal axis is activated [83]; consequently, several changes in blood constituents and reproductive efficiency are detected [6]. Thus, in this study, HS conditions have severe effects on serum sex hormones in experimental rabbit does. These drastic effects of HS conditions on the reproductive performance or sex hormones of females rabbits have been previously reported [14,84]. The female reproductive system is very sensitive to oxidative stress, and a reduction in the production and secretion of gonadotropins is necessary for the vital growth and development of ovarian follicles [11]. In this study, oral administration of BMHE at level 400 mg kg⁻¹ BW alleviated the harmful effects of HS on serum sex hormones in treated rabbit does above all other treatments. The progressive effects of maca on the reproductive performance and sex hormones of different experimental animals have been previously reported. Serum P4 levels increased in maca-treated female mice compared to the untreated group [85]. Similarly, Uchiyama et al. [86] demonstrated that maca intake enhances serum LH levels in female rats. Recently, El-Sheikh et al. [55] reported that maca-treated rabbit does have a numerically higher concentration of E2 hormone and a lower concentration of P4 hormone than those in the control group. This and the enhancement of serum LH levels may be the mechanisms that improve fertility [87]. The positive effects of maca may be due to saponins, which play an important role in sex hormone secretion and the treatment of sexual dysfunction. Generally, one of the maca’s main actions is stimulating the hypothalamus, which regulates the pituitary gland, acting as a tonic for the hormone system. Excess E2 levels can cause P4 levels to become too low, known as E2 dominance [88]. Administration of maca may help balance the E2/P4 ratio, leading to potential increased female fertility and achieving a healthy pregnancy in treated animals [89] by eliminating FR and generating an antioxidant function, mainly due to its metabolites (alkaloids and glycosylates) [90].

5. Conclusions

Based on the results, it could be concluded that oral administration of 400 mg kg⁻¹ BW of doe day⁻¹ for 1 week before matting is the suitable level of BMHE to alleviate HS conditions of V-line rabbit does. The promising and applicable effects of maca might improve productive and reproductive performance parameters alongside its serious role as an antioxidative agent and enhance the physiological and immune responses of HS rabbit does. Further studies are required to address the mechanisms of action and active principles of the maca plant or its extracts not only for rabbits but also for other economic farm animals, especially those reared in tropical, subtropical, and agroecological environments.