POTENTIAL OF Telfairia occidentalis LEAF EXTRACT AS BOAR

The preservation of semen for Artificial insemination (AI) required the use of extender, which served as a buffer media to keep the sperm cell in good condition for fertilization. In this study, fluted pumpkin Telfairia occidentalis leaf extract (TOLE) potential as an unconventional boar semen extender was compared to Beltsville thawing solution (BTS), a conventional semen extender over a 48hr storage period. Aqueous solution of TOLE was prepared at three (3) concentration levels (25, 50 and 75%). Fresh ejaculates from boar were extended at (1:4) in TOLE solutions and BTS extender in a completely randomized design; treatments were replicated four (4) times. Extended semen was stored at 17C and evaluated for semen qualities which include pH, dead/live ratio (%) and sperm cell concentration at time intervals (0, 12, 24, 36 and 48) hrs. Extended semen was significantly (P < 0.05) affected by semen extenders and storage periods for pH, and dead/live ratio (%) but sperm cell concentration (10/cm) not (P > 0.05) affected. The pH values for BTS was significantly (P < 0.05) higher as compared to TOLE extenders. 75% TOLE extender had highest value (43.89%), and BTS had lowest value (12.15%) for dead/live ratio significantly (P < 0.05). The pH and dead/live ratio (%) values increased over storage time in all extender. The optimum performance of TOLE as compared to BTS extender was recorded at 50% concentration of TOLE extender. TOLE showed a potential as boar semen extender, but further studies are required to validate and improve its application.


Introduction
Swine farming in sub-Saharan Africa is faced with challenges which include nutrition, health management and breeding. The need to sustain existence of good breeds has led to importation of pigs from other continents a procedure since replaced by artificial insemination (AI). An important aspect of AI that has facilitated its wide application is storage of semen for subsequent use.
Artificial insemination (AI) techniques enhance production rates and carcass homogeneity as well as the application of new management systems; hence have increased in the last decade. The advantage of AI is that the genetic potential of the best boar can be transferred to a large number of sows, leading to genetic improvement at a large scale (Kaeoket et al., 2010). An important aspect of AI that has facilitated its wide application is storage of semen for subsequent use. The seminal plasma supplies the necessary nutrients for the high metabolic demands of sperm transport through the female genital tract. In the ejaculate, this high metabolic activity can only be maintained over a limited period . As temperature declines, the proportion of spermatozoa that maintain normal membrane integrity, ultrastructure and biochemical components decreases (Johnson et al., 2006).
The important traits for quantitative and qualitative evaluation of ejaculate include: volume of ejaculate, concentration of sperm, motility, percentage of abnormal spermatozoa, total number of spermatozoa and number of dead/live spermatozoa (Savić et al., 2015(Savić et al., , 2017. Evaluation of sperm cell concentration, volume and percent of live spermatozoa is very important for the determination of maximal dilution of sperm suitable for artificial insemination or for a number of sows which can be inseminated (Savić et al., 2017, Ivelina 2017. The fertilizing ability of stored semen is achieved by using a semen extender, which is a liquid diluent added to the semen (Savić et al., 2017). This ensures that the functional characteristics of the sperm cells are maintained such that a higher conception rate is achieved (Gadea 2003). Factors affecting the properties of an extender include pH, ionic strength, ions and osmotic pressure of the medium. Anti-microbial substances are also commonly included in diluents (López Rodríguez et al., 2012). A number of extenders have been developed which decrease the metabolic activity of spermatozoa using an environment high in CO 2 at ambient temperature (Gadea 2003;Johnson et al., 2006;Boonkusol et al., 2010). Presently, Beltsville-TS (BTS) is one of the most widely used semen extender for both short and long term storage. However, with the increase in AI practices, sub-Saharan African countries are faced with the challenges of semen extenders availability, due to the high cost through importation. Therefore, there is need to find an alternative diluent for short term storage of boar semen.
Telfairia occidentalis (T. Occidentalis) (family cucurbitaceous) is a tropical vine grown in West and Central Africa. It is a popular vegetable in Nigeria, commonly called "Ugwu" and highly reputed in traditional medicine (Agatemor 2006;Fasuyi and Nonyerem 2007;Kayode and Kayode 2011). The plant produces luxuriant edible green leaves, which are rich in minerals, (Salman et al., 2008;Oboh et al., 2010), and vitamins (Kayode and Kayode 2011). The leaf also contains phytochemicals and has some antiplasmodial properties (Salman et al., 2008;Oboh et al., 2010;Kayode and Kayode 2011). Despite the need for affordable semen extenders in Nigeria and the proven rich nutritional profile of T. Occidentalis, studies on the usage of T. Occidentalis as a potential semen extender are lacking in Nigeria pig production industry. Therefore, the broad objective of this research is to investigate the potential of T. Occidentalis as a short-term extender for boar semen.

Location of study
The study was conducted at the Piggery unit, Teaching and Research Farm of the University of Ibadan, Nigeria. All analyses and examinations were carried out at the University Physiology Laboratory.

Processing of aqueous Telfairia occidentalis leaf extracts (TOLE)
Fresh leaves were harvested from matured stems at onset of flower emergence. Leaves were rinsed off debris with distilled water. T. Occidentalis leaves (350 g) were homogenized with distilled water using a homogenizer and the homogenate filtered with a Whatman filter paper (Oboh et al., 2010). Proximate analyses content were determined using the standard protocols (AOAC, 1995). The filtrate was then concentrated to one tenth of its original volume and stored at 4 o C in a refrigerator using an airtight plastic jar. At the point of application as semen extender, a serial dilution was carried out to have 25%, 50% and 70% concentrations.

Experimental animal and semen collection
Four seven-month-old boars were housed in individual pens, under the same environmental conditions, and fed a standard commercial diet to meet their nutrient requirements. Prior to semen collection, males were trained to mount a dummy sow and semen was collected using the hand gloved method. Ejaculates were collected once a week over the entire 6 weeks' training period. On the 7 th week, ejaculates were collected using the gloved-hand technique (Malo, 2010) into a thermos pre-warmed to 37 o C; lined with a disposable plastic bag (IMV International Corp., Maple Grove, MN); and covered with a disposable milk filter (IMV International, Eden Prairie, MN) . All ejaculates were pooled together and the volume of semen was estimated by weighing the ejaculate.

Extended semen analysis
Each ejaculate was diluted (1: 4) semen to extender respectively at room temperature (22 0 C ± 0.6 0 C). Evaluation of semen was carried out at time intervals (0, 12, 24, 36 and 48) hrs. The 0 hr evaluation was carried out at 10mins post semen addition to extenders. Dead/live (D/L) ratio was evaluated by using the eosin-nigrosin stain described by (Łukaszewicz et al., 2011). The pH was determined using the pH meter (Model pH 25) by dipping the pH electrode into the semen and the readings shown on digital scale were taken after 5mins. The concentration of sperm cells present in extended samples was determined by using the improved Neuber haemocytometer as described by (Bonato et al., 2014).

Statistical analysis
A two way analysis of variance (ANOVA) was performed using the fixed effect model. Bonferroni was used to test for the significance (P<0.05) of variance for all recorded and calculated data between different treatments, main effect of factors (semen extenders and storage period were considered) using model: Yijk = µ + Ti +Sj + eijk Where Yij = Individual observation µ = General mean Ti = Fixed effect of semen extender (i = 1…..4) Sj = Fixed effect of storage period (j = 0………48) eijk = Expected error

Results and Discussion
The results of the proximate composition, as presented in Table 1, showed that aqueous extract of T. Occidentalis leaf meal has a potential protein and carbohydrate source comparable with other conventional plant protein and energy sources (Salman et al., 2008). The relatively rich nutrient profile of the leaf extract Potential of Telfairia occidentalis leaf extract as … 53 is complemented with early age of the leaves as at the time of harvesting (Fasuyi and Nonyerem 2007;Salman et al., 2008). In Table 3, the effect of extenders on pH, dead/live (D/L) %, and sperm cell concentration (10 3 /cm 3 ) were presented. The pH and D/L (%) parameters were significantly (P < 0.05) affected by semen extenders. The pH of extended boar semen in BTS extender had the highest value (7.78) which was significantly (P < 0.05) different to the values obtained for TOLE extenders. A unique trend observed among the TOLE semen extenders was that pH decreases as inclusion level increase. However, 75% TOLE had the lowest pH value (6.30) which was significantly (P < 0.05) different to 50% (6.83) and 25% (6.95) inclusion levels. The D/L ratio (%) was significantly (P < 0.05) affected by semen extenders as shown in Table 3. As reported for D/L ratio (%), TOLE (75%) had the highest value (43.89%) which was significantly (P < 0.05) different as compared to other extenders. BTS had value (10.15%), which was significantly lower (P < 0.05) as compared to TOLE extenders. The sperm cell concentration (10 3 ) was not affected by the semen extenders during storage.
As shown in table 4, pH decreased significantly (P < 0.05) over storage time in all TOLE extender treatments, while BTS showed an earlier increase in pH before a decrease. The dead/live ratio (%) increased significantly (P < 0.05) over storage time for all semen extenders. The sperm cell concentration (10 3 ) was not affected by storage period as reported in table 4. The initial pH of TOLE was 6.20, BTS 7.52, while that of fresh boar ejaculate used in this study was 7.40. Therefore, the initial variation between the pH values of the extended semen in TOLE and BTS can be attributed to difference in initial pH of extenders. The high content of carbohydrate in the form of NFE may be responsible for the low pH in TOLE, while bicarbonate in BTS is responsible for the relatively alkaline pH. The earlier increase in pH of BTS extended semen can be attributed to instability of the extender. According to Kaeoket et al. (2010), the pH does not become stable from the start of dilution in water and that different extenders show a different pattern of pH change over time. In this study, the presence of a bicarbonate buffering system in the BTS extender explained earlier pH-rise. This increases the CO 2 in the media to reduce metabolic activities of sperm cell during storage (Vyt et al., 2004(Vyt et al., , 2007. The results of pH in this study are consistent with study of Kaeoket et al. (2010) who reported that the pH values of the extended semen increase with increased storage time. As observed in this study, the presence of glucose in each extender caused a considerable reduction of intracellular pH during storage. This intracellular pH reduction obviously reduced sperm cell metabolism and increase sperm cells survive during storage (Johnson et al., 2006). However, high content of glucose in form of NFE found in TOLE might have resulted into intracellular acidosis, which caused sperm cell distortion and resulted into dead sperm cells with increase concentration (Bonato et al., 2014).
As shown in table 4, the values of D/L ratio (%) increased in all extenders over time. The quantity of living sperm cell is a function of dead/live ratio. The high values of dead/live ratio of about 50% recorded in TOLE extenders may be attributed to bacterial infection. In most cases, the testicular tissue and accessory glands of the boar are bacteria-free, and bacterial contamination of the ejaculate occurs during the semen collection process (Martin Rillo et al., 1998). BTS used in this study contained an antibiotic (penicillin), which inhibits bacteria proliferation. The extender component (glucose) and the temperature at which semen doses are stored (15-16ºC) promote the growth of most Gram negative bacteria such as Escherichia coli, Salmonella spp and Pseudomonas spp (Gadea 2003).
In extended semen, bacterial contamination leads to a series of alterations including reduced sperm motility, sperm agglutination, an increased proportion of altered acrosomes, dead sperm cells and pH lowering to acidic levels 5.7 -6.4 (Althouse et al., 2000). However, the reduction in dead/live ratio for semen extender (TOLE 50%) as compared to other concentration can be attributed to presence of phytochemicals in the leaf extract in required proportion. TOLE was reported to show inhibitory effect to some microbes which include Escherichia coli, Pseudomonas aeroginosa, Proteus spp and Salmonella typhii (Kayode and Kayode, 2011). The high concentration of these phytochemicals in TOLE 75% may be toxic for the sperm cells and resulted to high dead/live ratio, while low concentration of phytochemicals at TOLE 25% can enhance bacteria proliferation and high dead/live ratio recorded as compared to TOLE 50%.
Sperm cell concentration was not significantly (P<0.05) affected by extender treatments. Although the value decreased slightly over time as shown in table 4. An equal concentration of sperm cell was randomly extended for each treatment at the onset of storage. The slight decrease in values of sperm cell over time may be attributed to dissolution of dead sperm cells in extended media (Martin Rillo et. al., 1998)

Conclusion
The BTS extender, a commercial short -term diluent used in this study performed better as compared to TOLE extenders. TOLE 50% concentration had the optimum performance within the TOLE concentrations. The study shows that TOLE has potential to serve as short -term semen extender, with best result in 50% dilution. However, further studies are required to effectively validate TOLE application as short term extender with increase in storage time and evaluation of other semen quality parameters.