Research Article

Supplementing Basal Diet with Sesbania Grandiflora Tablet (SGtab) and its Effect on Semen Quality of Kacang Buck

Lalu Ahmad Zaenuri*, Rodiah Rodiah, Adji Santoso Dradjat, Oscar Yanuarianto, I Wayan Lanus Sumadiasa , Lukman Hy

Faculty of Animal Science University of Mataram, Majapahit Street, No. 62, Mataram Lombok, 83125, West Nusa Tenggara, Indonesia.

Received | January 26, 2023; Accepted | February 20, 2023; Published | April 25, 2023

*Correspondence | Lalu Ahmad Zaenuri, Faculty of Animal Science University of Mataram, Majapahit Street, No. 62, Mataram Lombok, 83125, West Nusa Tenggara, Indonesia; Email: [email protected]

Citation | Zaenuri LA, Rodiah R, Dradjat AS, Yanuarianto O, Sumadiasa IWL, Lukman HY (2023). Supplementing basal diet with sesbania grandiflora tablet (SGtab) and its effect on semen quality of kacang buck.. Adv. Anim. Vet. Sci. 11(5): 843-851.

DOI | http://dx.doi.org/10.17582/journal.aavs/2023/11.5.843.851

ISSN (Online) | 2307-8316

 

 

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Inadequate nutritional supplies directly result in low livestock productivity and reproductivity (De Angelis et al., 2021). Recently, several researchers have extensively studied and developed feeding strategies to control and improve ruminants’ reproduction characteristics. The reproductive performance of Bali cows, for example, was affected significantly by the addition of calcium soap-soybean oil (Suharti et al., 2017). The spermatozoa quality of Etawah cross-breed bucks could also be improved by Urea Moringa Molasses Multinutrient Block (Syarifuddin et al., 2021). Further, it has been proven that supplementing mango seeds in Damascus goat bucks’ diets improved both the quality and quantity of semen production for breeding programs (El-Din et al., 2021). Moringa oleifera in the diet increased the quality of male rabbit semen (El-Kashef, 2022). Oral supplementation of selenium has been reported to improve buck spermatozoa motility and morphology (Lukusa and Kabuba, 2021). The positive impact of melatonin has also been reported to increase mass motility, individual progressive motility, spermatozoa viability, and acrosome integrity (Samir et al., 2020). In addition, Sesban hay had no negative effects on the semen characteristics (Mahgoub et al., 2022). Oral administration of 250 mg/kg crassocephalum crepidioides leaf extract for 14 consecutive days can significantly increase the spermatozoa quality of West African Dwarf Buck (Ajani et al., 2022).

Some researchers have also documented the relationship between nutrients and reproduction performance of mature male sheep (Murray et al., 1990). The oral supplementation of coenzyme Q10 (CoQ10) increased the semen quality of bucks during summer (El-Sherbiny et al., 2022). Food with higher energy and protein content improved the reproductive performance of Baktiary sheep (Kheradmand et al., 2006). Likewise, Zn supplementation improved the semen quantity and quality of Cashmere buck (Liu et al., 2020). However, none of these studies examined the effect of SGtab on Kacang Buck semen.

Thus, it is clear that the better the quality and quantity of the feed, the higher the reproductive performance of livestock. Notably, a diet with insufficient protein can reduce the quality and quantity of semen (Brown, 1994). The crude protein content of sesbania grandiflora is quite high, 23.65% (Kumar et al., 2017) and 22.61% (Nouman et al., 2013). Dahlanudin et al. (2001) reported that dry matter feed intake (g/head/d), dry matter digestibility (%), supply protein microbes (mg/W 0.75), and energy metabolism consumption of sesbania grandiflora were 1,070±86.0, 78±5.4, 979.3±222.8, 11.4±0.60, respectively.

The nutrient content of native grass is relatively low; however, the main goat feed for traditional farmers was native grass with no supplement. Wahyono et al. (2019) reported that the average crude protein content and digestibility of nine types of native grass were 3.48–7.60% and 49.39–66.68%, respectively. The feed quality affects the semen quantity and quality. Thus, the bucks fed with native grass as the main feed need to be supplemented to improve the quality of semen.

Furthermore, sesbania grandiflora enriched with mineral mix, rice bran, molasses, lime, and salt in the form of a tablet (SGtab) is easy to create and feed to goats. It also increases the natural mating ability of males, whose semen will be taken and processed into liquid or frozen form. Therefore, this study evaluated the effects of SGtab supplementation in basal diet, a cheap protein and energy resource, in improving the semen quality of the Kacang Buck.

MATERIALS AND METHODS

Animals’ Management, Diets, And Schedules

The animals used in this study have recommendations from the Animal Ethics Committee with Registration Number: 12/UN18.F2/EC/2022. Of 25 Kacang Bucks, 10 with the best libido and health, with average age and body weight 2.7±0.35 y and 28.4±2.4 kg, respectively, were selected for use in this study. The animals were allowed to acclimatize for 5 days prior to treatment. The research was conducted from June to July 2022 (dry season in Indonesia). The whole experiment concluded within 40 days – 5-day acclimatization and 30-day sampling periods. Semen was collected on days 10, 15, and 20 in the pre-SGtab period and then on days 25, 30, and 35 during the SGtab period. They received a daily diet of native grass equivalent to 10% of their body weight from day 1 to day 35 (Table 1). They received only native grass from day 1 to day 20. From day 21 to day 35, they received the same diet as days 1 to 20 plus SGtab, as mentioned in Table 1.

 

Table 1: Experimental schedule. Experimental schedule

Note	Days
	Pre-SGtab				during SGtab
Acclimatization
Native grass minus SGtab	1–5
Native grass plus SGtab		10	15	20	25	30	35
Semen sampling		1	2	3	4	5	6
Blood sampling			1				2

 

To meet the nutrient requirements, the SGtab supplement was prepared according to the standard nutrient requirement of goats described by the National Research Council (1985). Each buck received equal amounts of 42 g SGtab three times daily at approximately 07:00 h, 13.00 h, and 18:00 h before being offered basal feed. The amount of SGtab was 128 g/day/head (Table 2). The animals were housed in individual wooden-floored crates. Fresh water was available ad-libitum throughout the experimental time points. All bucks were inspected for general health, especially good libido before use in the study.

 

Table 2: Composition of SGtab

Supplement ingredient	SGtab composition (%)	Total per head (g)
Sesbania G flour	35	45
Rice bran	23	28
molasses	27	35
Lime stone	7	9
Salt	6	8
Mineral mix	2	3
Total	100	128

Note: The nutrient composition of 128 g SGtab was 10, 50, 3.2, and 1.0% of PK, TDN, Ca, and P, respectively.

Sesbania grandiflora leaves were sun-dried for 3 days until water content was at approximately 5% before being ground to a powder form. SGtab was made by weighing each ingredient according to the requirement. All materials – sesbania grandiflora leaf flour, rice bran, lime stone, mineral mix, molasses, and salt – were mixed evenly and put into the mold at a rate of 42 g/tablet.

Semen Collection And Evaluation

Semen was collected by a trained technician from 10 bucks using artificial vagina for 6 consecutive ejaculates at approximately 07.00 h, before feeding, at 120-h interval from each Kacang Buck (Table 1). Collected semen was transferred as soon as possible to the laboratory and held in a 35°C water bath before further evaluation. Semen was evaluated for volume, pH, spermatozoa concentration/ml and concentration/ejaculates, plasma membrane integrity, viability, progressive motility, and morphology including normal and abnormal spermatozoa (no head, double head, no tail, and looped tail).

Briefly, semen volume was measured directly from notation on a collector tube. pH was assessed by a digital pH meter. Spermatozoa concentration/ml (109/ml) was counted by Neubauer hemocytometer, as described by Mahmoud et al. (2013). The examination of the plasma membrane integrity of the spermatozoa was carried out as explained by Susilawati (2011), as follows. First, 1 ml of hypo-osmotic solution of 150 m osmol (7.35 g sodium citrate, 2H2O, 13.52 g fructose dissolved in 1000 ml aquabides) was dropped into a test tube. Second, 0.1 ml semen was dripped into the test tube containing the hypo-osmotic solution and incubated at 37°C for 40 min. After incubation, one drop of the semen sample was placed on the object glass and examined using a light microscope with a magnification of 400×. The change expected to occur is the presence of swelling or circular tail. Two hundred spermatozoa are counted for the characteristic tail (circular) that identifies plasma integrity (Ahmad et al., 2003; Jeyendran et al., 1984; Susilawati, 2011). To evaluate normal spermatozoa morphology and viability, one drop of the semen sample and two drops of Nigrosin-Eosin were mixed in a warm object glass and left for 30 s. Next, a smear was made and dried on a warm plate. The percentages of normal morphology and viability were examined subjectively using a phase contrast microscope (Olympus CX 43, Optical co., Ltd., Tokyo, Japan) at 400 magnification connected to a monitor (Abdelnour et al., 2020; Marin et al., 2021; Susilawati, 2011). The spermatozoa with no head, double head, no tail, and looped tail were counted and calculated as abnormal spermatozoa. If the spermatozoa absorb color, they are unviable, and if they are transparent or do not absorb color, they are viable (Ibrahim et al., 2021; Jeyendran et al., 1984).

Progressively motile spermatozoa were estimated as a percentage. One drop of fresh semen was diluted in 20 ml NaCl (1:20; v/v) and then homogenized. One drop of the semen sample was placed on a 37°C glass slide with a cover slide and evaluated under a phase-contrast microscope connected to a monitor. The spermatozoa that moved forward were noted as progressively motile (Ibrahim et al., 2021; Marin et al., 1997).

Blood Sampling

Blood sampling was performed at 07.00 h, 15 days after acclimatization and before SGtab supplementation and on day 35, or 10 days after SGtab supplementation. Blood samples were taken in the morning before feeding according to Suharti et al. (2017), El-Din (2021), and Mahgoub et al. (2022) by jugular venipuncture into heparinized tubes to obtain blood serum. Blood samples (5 ml) were collected from four random bucks before and during SGtab supplementation. Blood samples were then centrifuged at 3500 g for 10 min for serum collection for subsequent analyses. Blood serum was measured for blood glucose, total protein, cholesterol, and ureum. The blood samples were analyzed at Hepatica Laboratory, Mataram, Lombok, West Nusa Tenggara Province, Indonesia.

Statistical Analysis

Pre- and post-test analyses were employed using T-test with repeated measuring; independent sample T-test was applied (SAS, 2022). A probability of less than 5% was considered significant. Data are expressed as Means ± SD and Standard Error Means (SEM).

RESULTS AND DISCUSSION

This study found that the semen volume of the Kacang Buck significantly increased when they were offered SGtab (Table 3). Similar results were reported in the bucks offered digitata leaves (Ibrahim et al., 2021). The inclusion of 15% mango seeds in the basal feed of Damascus goat bucks had a positive effect and improved semen production and quality in breeding programs (El-Din et al., 2021). Moringa leaves in urea molasses multi-mineral block did not increase the volume of the semen in Peranakan Etawah (PE) bucks (Syarifuddin et al., 2021). Some studies reported the normal semen volume of the Kacang Buck as 1 ml (Kusumawati et al., 2017) and 0.74±0.13 (Tungujama et al., 2021) and those of the Etawah, Bligon, and Kejebong bucks as 0.87 ml, 0.54 ml, and 0.63 ml, respectively (Rahmawati et al., 2014). The semen volume of bucks significantly increased with an additional intake of 40 mg zinc/kg dry matter (DM) (Liu et al., 2020). Semen volume per ejaculate depends on age, breed, feed quality, body size, and frequency of ejaculation, physical condition, and collector skills (Lemma and Shemsu, 2015).

No significant change was noted in the pH value of semen

 

Table 3: Effect of SGtab supplementation on semen volume, pH, and spermatozoa concentration of Kacang Buck

Variables	Average percentages (n=10)		SEM
	Before SGtab supplementation	During SGtab supplementation
Semen Volume	0.82±0.26a	1.12±0.37b	0.05
pH	6.99±0.28a	7.04±0.31a	0.04
Spermatozoa concentration (106/ml)	2,807±417.40a	2,955±384.11a	0.05
Spermatozoa concentration/ejaculate (×106)	2,319±0.84a	3,107±1.212b	0.14

Note: Different superscript in the same row differ significantly (P<0.05)

 

Table 4: Effect of SGtab supplementation on the percentages of plasma membrane integrity, spermatozoa viability and spermatozoa progressive motility, and integrity of plasma membrane (IPM) on Kacang Buck semen (n=10)

Variables	Before SGtab supplementation	During SGtab supplementation	SEM
Plasma membrane integrity	80.57±3.10a	83.80±3.33b	0.44
Spermatozoa viability	76.03±4.57a	80.167±2.97b	0.56
Progressively motile spermatozoa	74.53±2.97a	77.96±2.71b	0.51
IPM	74.11±4.78a	79.23±5.45b	0.74

Note: Different superscript in the same row differ significantly (P<0.05)

before and during SGtab supplementation. Significantly higher or lower pH causes low spermatozoa viability. The normal pH of semen in this study was in the range of 6.4–6.8 (Sekosi et al., 2016) and 6.5±0.34 (Tungujama et al., 2021).

Spermatozoa concentration/ml tends to be higher during SGtab supplementation compared to before supplementation; however, it was not significantly higher. As a result of higher semen volume and concentration per ml, the concentration of spermatozoa per ejaculate was significantly higher during SGtab supplementation compared to before supplementation (Table 3). This result shows a trend similar to those of previous studies wherein bucks supplemented with 40 mg zinc/kg DM showed increased spermatozoa concentration from 4.09 × 109/ml to 4.59 × 109/ml (Liu et al., 2020). Kusumawati et al. (2017) reported a concentration of 3.540 × 106/ml. However, a contrary result was reported by Thasmi et al. (2022) that showed that continuous additional intake of the extract of etanolik malaka leaf for 21 days tended to decrease spermatozoa in rats.

Host test in this study found that the percentages of spermatozoa with a normal membrane were significantly higher in bucks during SGtab supplementation compared to no supplementation (Table 4). It is well known that plasma membrane integrity is an essential requirement for normal functioning of spermatozoa. Therefore, the more spermatozoa with normal membrane the higher will be the percentage of good-quality spermatozoa (Makarevich et al., 2011).

The percentages of spermatozoa cell livability of Kacang Bucks with SGtab supplementation were significantly higher than those with no supplementation (Table 4). Spermatozoa livability in WAD bucks increased from 96.40±1.60 before supplementation with crassocephalum crepidioides to 98±00 and 98.00±00 after being supplemented, respectively, Ajani et al. (2022). Lower spermatozoa livability (61.58±8.50% and 95.8%, respectively) was reported by Tunggujana et al. (2021) and Kusumawati et al. (2017).

The percentages of progressively motile spermatozoa are one of the vital indicators of good-quality semen in both animals and humans (Abdelnour et al., 2019). This research found that progressively motile spermatozoa significantly increased in the bucks supplemented with SGtab compared to those not supplemented with it (Table 4).

This study proved that the supplementation of SGtab in basal Kacang Buck diets can improve the quality of semen produced compared to those not supplemented with SGtab. Several similar studies have shown relatively similar results. Digitata leaf feeding is reported to have a positive impact on semen quality (Ibrahim et al., 2021). Oral supplementation of Coenzyme Q10 at a dose of 3 mg/kg increased the percentage of progressive motile spermatozoa from 69.00±1.77 at week 0 to 78.00±2.14 at week 5 after supplementation with CoQ10 (El-Sherbiny et al., 2022). Likewise, the percentage of viability, normal morphology, and spermatozoa concentration increased significantly from 82.40 ± 1.1, 83.60±2.66, 2.22±0.02×109/ml in week 1 to 91.60±1.44, 91.40±0.54 and 2.45±0.01×109 in week 6, respectively (El-Sherbiny et al., 2022). Oral administration of crassocephalum crepidioides extract was also shown to increase the percentage of spermatozoa motility in WAD bucks from 87.00±4.06 on day 0 to 95.00±0.00 and 96.50±1.50 on days 7 and 14, respectively (Ajani et al., 2022). The percentage of spermatozoa progressive motility in thawed semen of Saanen bucks that received oral

 

Table 5: Percentages of normal and abnormal spermatozoa as an effect of SGtab supplementation

Variables	Before SGtab supplementation	During SGtab supplementation	SEM
Normal	88.97±2.93a	92.47±2.86b	0.44
Abnormal	11.03±2.93a	7.53±2.86b	0.43
No head	2.37±1.19a	1.47±1.33b	0.17
Double head	2.53±1.68a	1.83±1.23a	0.19
No tail	3.03±0.96a	2.13±1.19b	0.15
Looped tail	3.10±1.37a	2.10±1.24b	0.18

Note: Different superscript in the same row differ significantly (P<0.05)

 

Table 6: Result of blood sample analysis of bucks before and during supplementation with SGtab

Variables	Before SGtab	during SGtab	SEM
Glucose	47.75±4.65a	56.25±6.45a	2.44
Total protein	5.7±0.22a	6.05±0.37a	0.11
Cholesterol	46.5±4.65a	43.75±8.22a	2.24
Ureum	19.75±2.63a	20.75±3.3a	0.99

Note: Means in the same row with different superscript differ significantly (P<0.05)

supplementation of selenium was 41.01±0.61, which was significantly higher compared to that of bucks with no selenium supplementation (37.11±1.63) (Lukusa and Kabuba, 2021).

This study found that the percentages of IPM are much higher in the spermatozoa of bucks offered SGtab than those who were not offered supplements (Table 4). Normal plasma membrane integrity of spermatozoa is an important requirement for normal metabolism. Thus, a high percentage of spermatozoa with normal membrane is necessary for frozen semen (Makarevich et al., 2011). The percentage of spermatozoa with an intact membrane of Saanen bucks supplemented with selenium was 42.01±3.02 compared to 35.04±2.12 in bucks that were not supplemented (Lukusa and Kabuba, 2021).

This study also found that the percentage of abnormal spermatozoa significantly decreased from 11.03±2.93 to 7.53±2.86 before and during SGtab supplementation, respectively (Table 5). The percentage of abnormal spermatozoa found in this study was in line with the results reported in other studies as follows. The supplementation of 40 mg zinc/kg DM feed decreased the percentage of abnormal spermatozoa from 15.1 to 14.4 (Liu et al., 2020). A low percentage (2.9%) was reported by Kusumawati et al. (2017) and a higher percentage (14.7±0.00) was reported by Oyeyemi et al. (2021). The percentages of abnormal spermatozoa should not more than 15% (Saili et al., 2016) to 20% (Sekosi et al., 2016). Semen quality is considered substandard if the percentage of abnormal spermatozoa is higher than 20% (Arifiantini and Purwantara, 2010). Thus, semen quality from this study is acceptable because the percentage of abnormal spermatozoa was lower than 15% (Table 5). Standard percentages of buck spermatozoa abnormalities suitable for artificial insemination is not more than 15% (Ax at al., 2008) or 20% (Karatasudjana, 2001). Ax et al. (2008), in addition, stated that fertility decreases when the percentage of abnormal spermatozoa is more than 25% of the total spermatozoa.

Buck blood serum parameters before and during supplementation were presented in Table 6. This research found that the addition of SGtab increased the concentration of blood glucose, total protein, and ureum; however, they were not significant. The concentration of cholesterol, on the other hand, was slightly decreased in the bucks after supplementation with SGtab compared to those before.

The percentage of motile sperm in Tris-citrat extender enriched with cholesterol at 0.5 mg/ml was higher compared to either 0.0 or 1.0 mg/ml, but the difference was not significant. However, the average percentages of motile spermatozoa after thawing was significantly higher in extender enriched with 1 mg/ml (56.0%) compared to 0.0 mg/ml (47.5%) and 0.5 mg/ml (51.5%), respectively (Sitomorang, 2002). In contrast, the low cholesterol content in semen plasma and spermatozoa causes the susceptibility of spermatozoa to cold shock (White, 1993). Cholesterol, in addition, is an important factor to maintain membrane integrity (Voet and Voet, 1990) and the addition of cholesterol to semen extender is expected to increase the viability of spermatozoa, which will eventually increase the percentage rate of pregnancy.

Two studies reported a significant increase in the total blood protein of chickens when the feed was supplemented with moringa oleifera leaf (Voemesse et al., 2018) and rabbit (El-Kashef, 2022). However, in contrast with the results of this study, two others studies reported the effect of proteins on reproductive parameters of rams, where protein supply above normal needs had no effect on reproductive parameters, such as testicular size, semen quality, testosterone secretion, or sexual activity (Fernandez et al., 2004; Bielli et al., 1999). Singh et al. (2018) report that optimal feed and nutrition management will have an impact on reproductive health and gonadotropin secretion. This study also found an insignificant increase in blood glucose after SGtab supplementation. Dance et al. (2016) and Geary et al. (2016) report that the level of energy, protein, minerals, and vitamins at every stage of livestock growth and reproduction is very important to maximize the fertility rate of cattle. They also state that the quality of spermatozoa and spermatogenesis is improved if the balance of energy and protein is met through mineral supplementation during the pre-pubertal period.

Protein is required by almost all living things not only for maintenance but also because it carries vitamins and hormones and regulates the metabolism of the entire body (Anderson and Anderson, 2002). When serum proteins change, it may be a potential diagnostic indication of some pathological process. Therefore, serum protein could determine the physiological condition of livestock to differentiate between healthy and unhealthy animals (Tothova et al., 2016).

The values obtained from the proximate analyses of SGtab (percentages), such as water, ash, crude fat, crude fiber, and crude protein, were 19.70, 18.80, 0.34, 7.52, and 6.54, respectively. In this study, some nutritional content of SGtab may have had a significant effect on the semen quantity and quality of the Kacang Buck. Na, K, Cl, Ca, Mg, P, S are essential minerals in the semen with important functions (Marzec-Wróblewska et al., 2012). In addition, such mineral requirements need to be met for spermatogenesis, the promotion of motility, and the quality of spermatozoa, as well as for the development of Sertoli and Leydig cells (Tvrdá et al., 2013).

Two studies report that minerals play a significant role to promote spermatozoa quality, such as progressive, rapid, and velocity in buck spermatozoa (Arangasamy et al., 2018; Liu et al., 2019). Similar results were reported in sheep and cattle (Kumar et al., 2017; Ghorbani et al., 2018). The result of this research is similar to that of a study where Red Sokoto bucks were fed with whole cottonseed (Itodo et al., 2021), in which the plant has high mineral content, such as potassium (Namratha and Sahithi 2015). Potassium is an essential mineral to maintain semen volume (Tvrdá et al., 2013). The same result is reported by Liu et al. (2020); the addition of a small amount of Zn and Cu in feed linearly improved the quantity and quality of semen of Cashmere goats. Increase in parameters such as semen volume, mass motility, viability, membrane integrity, acrosome integrity, and progressively motile spermatozoa was also noted (Mayasula et al., 2021). Therefore, the supplementation of mineral in feed improved semen volume and quality (Arangasamy et al., 2018). However, this research did not analyze SGtab mineral content. Nevertheless, SGtab was composed of lime stone, salt, and mineral mix at 7, 6, and 2% of the total SGtab mixtures, respectively (Table 2).

Since there is no reference pertaining to the effect of native grass as a single feed on buck semen quality, this study has proven that the addition of SGtab enriched with various micro-minerals to native grass-based feed increases nutritional supply and thus increases the production and quality of Kacang Buck spermatozoa. The results of the analyses of blood samples proved that the values of SGtab, glucose, total protein, and ureum were higher than those before supplementation with SGtab (Table 6).

CONCLUSION

The whole parameter of semen quality increased in Kacang buck offered native grass basal diet supplemented with Sesbania grandiflora tabtlet (SGtab). Further researches is recommended to evaluate the effects of different quantity of SGtab on semen quality.

AKNOwLEDGeMENT

The authors are thankful to the Research Project Unit of the Mataram University who has financed this research.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interests

NOVELTY STATEMENT

Sesbania grandiflora tree is a common three in tropical country, rich protein and easy growing. It could also be used as a very high quality feed supplement after enriching with some ingredients such as mineral, molasses, salt and lime stone. This study proven that offering SGtab into basal diet could increase quality and quantity of Kacang Buck semen better than non-supplemented buck.

AUTHORS CONTRIBUTION

All authors were involved in all proses of this study since the draft of proposal to the final report and the final draft of manuscript to be submitted in the journal.

REFERENCES

Abdelnour SA, Abd El-Hack M.E, Alagawany M, Taha AE, Elnesr SS, Elmonem OMA, Swelum AA (2020). Useful impacts of royal jelly on reproductive sides, fertility rate and spermatozoa traits of animals. J. Anim. Physiol. Anim. Nutr. 104:1798–1808. https://doi.org/10.1111/jpn.13303

Ahmad, Z., M. Anzar, M. Shahab, N. Ahmad and S.M.H. Andrabi. (2003). Sephadex and sephadex ionexchange filtration improves the quality and freezability of low-grade buffalo semen ejaculates. Theriogenology. 59: 1189-1202

Ajani OS, Oyeyemi MO, Wahab AT (2022). Crassocephalum crepidioides improves semen quality and gonadometric indices in the West African dwarf bucks. Nig. J. Anim. Prod. 49(1): 60-66. https://doi.org/10.51791/njap.v49i1.3400

Anderson NL, Anderson NG (2002). The human plasma proteome. Molecul. Cellul. Proteom. (1): 845–867.

Arangasamy A, Krishnaiah MV, Manohar N, Selvaraju S, Guvvala PR, Soren NM, Reddy IJ, Roy KS, Ravindra JP (2018). Advancement of puberty and enhancement of seminal characteristics by supplementation of trace minerals to bucks, Theriogenology., (110): 182–191. https://doi.org/10.1016/j.theriogenology.2018.01.008.

Arifiantini R, Purwantara B (2010). Motility and Viability of Friesian Holstein Spermatozoa In Three Different Extender Stored At 5oC. Journal of the Indonesian Trop. Anim. Agricult., 35(4): 222-226. https://doi.org/10.14710/jitaa.35.4.222-226

Ax R L, Dally M R,.Didion BA, Lenz RW, Love CC, Varner DD, Hafez B, Bellin ME (2008). Artifical Ainsemination. In: Hafez E.S.E and Hafez B., editors. Reproduction In Farm Animals 7ed. Australia Blackwell Publishing. pp: 365-375.

Bielli A, Pedrana G, Gastel MT, Castrillejo A, Morana A, Lundeheim N, Forsberg M., Rodriguez-Martinez H (1999). Influence of grazing management on the seasonal changes in testicular morphology in Corriedale rams. Anim. Reprod. Sci. 56, 93-105. https://doi.org/10.1016/S0378-4320(99)00027-5

Brown BW (1994). A review of nutritional influences on reproduction in boars, bulls and rams. Reprod. Nutr. Dev., 34: 89-114. https://doi.org/10.1051/rnd:19940201

Dahlanuddin, Zaenuri LA, Mashur, dan Muzani M (2001). Optimalisasi Penggunaan Daun Turi (Sesbania grandiflora) sebagai Pakan Ternak Kambing. Fakultas Peternakan Universitas Mataram dan Balai Pengkajian Teknologi Pertanian (BPTP) NTB.

Dance A, Thundathil J, Blondin P, Kastelic J (2016). Enhanced early-life nutrition of Holstein bulls increases sperm production potential without decreasing postpubertal semen quality. Theriogenology, 86(3): 687-694.e2. https://doi.org/10.1016/j.theriogenolog y.2016.02.022

De Angelis A, Gasco L, Parisi G, Danieli PP (2021). A Multipurpose Leguminous Plant for the Mediterranean Countries: Leucaena leucocephala as an Alternative Protein Source: A Review. Animals. 11: 2230. https://doi.org/10.3390/ani1108223

El-Din SAM, El-Esawy GS, and El-Sanafawy HA (2021). Effect of Dietary Partial Replacement of Corn Grains by Mango Seeds on Productive and Reproductive Characterization of Damascus Goat Bucks at Prepubertal Stage. J. Anim. Poult. Prod., Mansoura Univ., 12 (2):61 – 69. https://doi.org/10.21608/jappmu.2021.153289

El-kashef MMA (2022). Effect of moringa olivera leaves on physiological response, hormone changes and quality of male rabbit under north Sinai conditions. Egyptian J. Rabbit Sci., 32(2): 105- 119

El-Sherbiny HR, Abdelnaby EA, El-Shahat KH, Salem N, Ramadan ES, Yehia S., Fathi M. (2022). Coenzyme Q10 Supplementation enhances testicular volume and hemodynamics, reproductive hormones, spermatozoa quality, and seminal antioxidant capacity in goat bucks under summer hot humid conditions. Vet. Res. Commun. (2022) 46:1245–1257. https://doi.org/10.1007/s11259-022-09991-8

Fernandez M, Giralde FJ, Frutos P, Lavin P, Mantecon AR (2004): Effect of undegradable protein supply on testicular size, spermiogram parameters and sexual behavior of mature Assaf rams. Theriogenology (62): 299-310.. https://doi.org/10.1016/j.theriogenology.2003.10.003

Geary TW, Kelly WL. Spickard DS, Larson CK, Grings EE, Ansotegui R P (2016). Effect of supplemental trace mineral level and form on peripubertal bulls. Anim. Reprod. Sci., (168): 1–9. https://doi.org/10.1016/j.anireprosci.2 016.02.018

Ghorbani A, Moeini MM, Souri M, Hajarian H (2018). Influences of dietary selenium, zinc and their combination on semen characteristics and testosterone concentration in mature rams during breeding season, J. Appl. Anim. Res., 46: 813–819. https://doi.org/10.1080/09712119.2017.1406858

Ibrahim K, Umaru MA, Jibril A, Umar AA, Adeyeye AA, Stephen J (2021). Effects of graded levels of Adansonia digitata leaves on semen quality in Red Sokoto bucks. Comparat. Clin. Pathol., 30: 821–828. https://doi.org/10.1007/s00580-021-03283-4.

Itodo JI, Ibrahim RP, Rwuaan JS, Aluwong T, Shiradiyi BJ, Owoicho AK, Azubuike US, Agbi KA (2021). The effects of feeding graded levels of whole cottonseed on semen characteristics and testicular profiles of Red Sokoto Bucks. Acta Scientiarum Anim. Sci. 30:43. https://doi.org/10.4025/actascianimsci.v43i1.50990

Jeyendran R. S., H.H. Van Der Ven, M. P. Pelaez, B. G. Crabo, L.J.D. Zaneveld. (1984). Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Fertil. 70: 219-228

Kartasudjana R (2001). Ciri-ciri atau Tanda Keabnormalitasan pada Semen Kambing Peranakan Etawah. dalam Seminar Nasional Hasil Penelitian.

Kheradmand A, Babaei H, Batavani RA (2006). Effect of improved diet on semen quality and scrotal circumference in the ram. Veterinarski Arhiv., 76 (4): 333-341,

Kumar U, Murthy HNN, Singh KC, Gouri MD, Rajeshwari YB, Siddeshwara NC, Mateen A, Guruprasad R (2017). Biomass yield and chemical composition os sesbania grandiflora and moringa oleifera. Int. J. Sci. Environ. Technol., 6(6): 3264 – 3269.

Kusumawati ED, Utomo KN, Nugroho AT, Krisnaningsih dan Rahadi S (2017). Kualitas semen kambing kacang dengan lama seimpan yang berbeda pada suhuruang penggunakan pengencer tris aminomethan kuning telur. JITRO., 4(3): 42-51. https://doi.org/10.33772/jitro.v4i3.3894

Lemma A, Shemsu T (2015). Effect of age and breed on semen quality and breeding soundness evaluation of preservice young bulls. J. Reprod. Infertil. 6 (2): 35-40. https://doi.org/10.5829/idosi.jri.2015.6.2.94131

Liu H, Yabo S, Zhao N, Dong W, Yang G (2019). Effect of Zinc Supplementation on Semen Quality, Spermatozoa Antioxidant Ability, and Seminal and Blood Plasma Mineral Profiles in Cashmere Goats. Biolog. Trace Element Res., 196:438–44. https://doi.org/10.1007/s12011-019-01933-x

Lukusa K, Kabuba J (2021). Oral supplementation of selenium improves post-thaw spermatozoa quality in Saanen bucks. Asian Pacific J. Reprod. 10(4): 187-194. https://doi.org/10.4103/2305-0500.321126

Mahgoub AAS, El-Hafeez AMA, Mohamed MY, Shaarawy A-MBM, Nassar MI. (2022). Efficiency of feeding sesban hay as a replacement for clover hay on growth performance and semen quality of sheep. Adv. Anim. Vet. Sci. 10(7): 1434-1443. https://dx.doi.org/10.17582/journal.aavs/2022/10.7.1434.1443

Mahmoud GB, Abdel-Raheem SM, Hussein HA (2013). Effect of combination of vitamin E and selenium injections on reproductive performance and blood parameters of Ossimi rams. Small Rumin. Res. 113: 103–108. https://doi.org/10.1016/j.smallrumres.2012.12.006

Makarevich AV, Spalekova E, Olexikova L. Lukac N, Kubovicova E, Hegedusova Z. (2011). Functional characteristics of ram cooling-stored spermatozoa under the influence of epidermal growth factor. Gen. Physiol. Biophys. 30: 36–43.

Marin GJ, Mahan DC, Chung YK, Pate JL, Pope WF (1997). Effects of dietary selenium and vitamin E on boar performance and tissue responses, semen quality and subsequent fertilization rates in mature gilts. J. Anim. Sci. 75:2994–3003. https://doi.org/ 10.2527/1997.75112994x

Marzec-Wroblewska U, Kaminski P, Łakota P (2012). Influence of chemical elements on mammalian spermatozoa. Folia Biologica. (Praha), 58: 7-15.

Mayasula VK, Arunachalam A, Babatunde SA, Naidu SJ, Sellappana S, Krishnan BB, Rajendran US, Janardhan RI, Bhatta R. (2021). Trace minerals for improved performance: a review of Zn and Cu supplementation effects on male reproduction in goats. Trop. Anim. Health Prod. 53: 491. https://doi.org/10.1007/s11250-021-02943-5

Murray PJ,. Rowe JB, Pethic DW, Adams NR (1990). The effect of nutrition on testicular growth in the Merino ram. Aust. J. Agric. Res. 41: 185-195. http://dx.doi.org/10.1071/AR9900185

Namratha V, Sahithi P (2015). Baobab: a review about “The Tree of Life.” Int. J. Adv. Herbal Sci. Tech., 1(1):b20.

National Reseacrh Council (NRC). (2007). Nutrient Requirement of Small Ruminant (Sheep, Goat, Cervids, and New World Camelids). The National Academies Press, Washington, D.C

Nouman W, Basra SMA, Siddiqui MT, Yasmeen A, Gull T, Cervantes MC, Alcayde. (2014). Potential of Moringa oleifera L. as livestock fodder crop: a review. Turk. J. Agric. For.38: 1-14. https://doi.org/10.3906/tar-1211-66

Oyeyemi MO, Olukole SG, Ajayi TA, Adeniji DA. (2011). Semen characteristics and sperm morphological studies of the West African dwarf buck treated with Aloe vera gel extract. Iranian Int. J. Reprod. Med. 9(2):83–88

Syarifuddin NA, Rizal M, Riyadhi M, Wahdi A (2021). Semen Quality of Etawah Crossbreed Bucks Fed with Urea Moringa Molasses Multinutrient Block Supplemented with Native Grass Basal Feed. Adv. Biolog. Sci. Res., 17: 40-48

Rachmawati L, Ismaya, Astuti P (2014). Korelasi antara hormon testosteron, libido, dan kualitas spermatozoaa pada kambing bligon, kejobong, dan peranakan etawah. Buletin Peternakan. 38(1): 8-15.

Saili T, Rahadi S, Sura IW, Lopulalan F (2016). Sinkronisasi Estrus dan Inseminasi Buatan Menggunakan Semen Cair Hasil Sexing pada Sapi Bali Induk Yang Dipelihara dengan Sistem yang Berbeda. J. Ilmu Ternak. 16(2):49-55. https://doi.org/10.24198/jit.v16i2.11576

Samir H, Nyametease P, Elbadawy M, Naggaoka K, Sasaki K, Watanabe G (2020). Administration of melantonin improves testicular blood flow, circulatinghormones , and semen quality in Shiba goats. Theriogenology. 146: 111-119. https://doi.org/10.1016/j-theriogenology.2020.01.053

SAS (2002). SAS/STAT User’s Guide. Release 8.1. Statistical Analysis System. SAS Institute Inc., Cary, NC, USA.

Sekosi PPP, Kusumawati ED, dan Krisnaningsih ATN (2016) Motilitas dan Viabilitas Semen Segar Kambing Peranakan Etawa (PE) dengan Menggunakan Pengencer Cauda Epididymal Plasma (CEP-2) pada Lama dan Suhu Simpan yang Berbeda. J. Sains Peternakan. 4(1): 34-49.

Singh AK, Rajak S, Kumar P, Shilpi KYR (2018). Nutrition and bull fertility: A review. J. Entomol. Zool. Stud., 6(6): 635–643.

Sitomorang P (2002). Pengaruh Kolesterol Terhadap Daya Hidup dan Fertilitas Spermatozoa Sapi. JITV., 7(4): 251-258

Suharti S, Khotijah L, Nasution AR, Warmadewi DA, Cakra GLO, Arman C, Wiryawan KG (2017). Productive and reproductive performances and blood profile of Bali cows supplemented with calcium soap-soybean oil. Pak. J. Nutr., 16: 882-887. https://doi.org/10.3923/pjn.2017.882.887.

Susilawati T (2011). Spermatozoaatologi. Universitas Brawijaya Press (UB Press), Malang (Indonesia).

Thasmi CN, Asmilia N, Suryani ES, Hafizuddin H, Adam M, Sayuti A, Nazaruddin N, Panjaitan B. (2022). Effect of ethanol extract from Malacca leaves (Phyllanthus emblica) onn the sperm quality of mice. Jurnal Kedokteran Hewan. 16(3):106-109. https://doi.org/10.21157/j.ked.hewan.v16i3.27246

Tothova C, Nagy O, Kovac G. (2016). Serum proteins and their diagnostic utility in veterinary medicine: a review. Vet. Med., 61(9): 475–496. https://doi.org/10.17221/19/2016-VETMED

Tunggujama OU, Kaka A, dan Pati DU (2021). Karakteristik dn kualitas semen kambing kacang dalam pengencer tris kuning telur yng disuplementasi dun kelor (Moringa Oleifera). J. Peternakan Sabana, 1(2):70-74. https://ojs.unkriswina.ac.id/index.php/sabana

Tvrdá E, Sikeli P, Lukáčová J, Massányi P, Lukáč N (2013). Mineral nutrients and male fertility. J. Microbiol. Biotechnol. Food Sci., 3(1):1–14

Venkata KM, Arunachalam A, Babatunde SA, Naidu SB, Sellappana S, Krishnan BB, Rajendran US, Janardhan RI, Bhatta R (2021). Trace minerals for improved performance: a review of Zn and Cu supplementation effects on male reproduction in goats. Trop. Anim. Health Prod., 53: 491. https://doi.org/10.1007/s11250-021-02943-5

Voemesse K, Teteh A, Nideou D, N’nanlé O, Gbeassor M, Decuypere E, Tona K (2018). Effect of Moringa oleifera leaf meal on growth performance and blood parameters of egg type Chicken during Juvenile growth. Int. J. Poult. Sci., 17(4): 154-159. https://doi.org/10.3923/ijps.2018.154.159

Voet D, Voet JG (1990). Biochemistry. John Wiley and Sonc, Inc. Canada. pp. 278-279.

Wahyono TE, Jatmiko, Firsoni SN, Hardani W, Yunita E (2019). Evaluasi Nutrien dan Kecernaan In Vitro Beberapa Spesies Rumput Lapangan Tropis di Indonesia. Sains Peternakan. 17 (2):17-23. http://dx.doi.org/10.20961/sainspet.

White IG (1993). Lipids and calcium uptake of sperm in relation to cold and preservation: a review. J. Reprod. Fertil. Dev. 5:639-658.