The Effect of Liquid Fermented Potato Hash Diet on Testicular Size, Weight and Epididymal Semen Quality of Large White × Landrace Boars

Abstract

The study aimed to evaluate testicular size, weight and epididymal semen quality of Large White × Landrace (LW × LR) boars fed fermented liquid potato hash. Diets containing either 200 (LFLPH) g/kg potato hash; 400 (HFLPH) g/kg potato hash or no fermented potato hash (control). Forty-two crossbred boars (LW × LR) weighing (25 ± 2.3 kg) were individually housed and fed ad-lib one of the seven dietary treatments for three months. Pigs were allocated to diets in complete randomized design. After 3 months and 600 ± 4 kg average body weight boars were slaughtered and epididymal semen was collected from head of epididymis. Boars that were fed control had higher (P < 0.05) live spermatozoa concentration than LLFPH and HFLPH diets. However, HLFPH had higher (P < 0.05) proximal droplet, distal droplet and dead spermatozoa concentration than control and LLFLPH. In addition, boars that were fed control and LLFPH had higher (P < 0.05) total, progressive, rapid motility, and lower non-progressive, static, medium and slow motility than HLFPH. It is concluded that low liquid fermented potato hash diet could be used as an alternative feed source for pigs. The results indicated that diets contain LFLPH can be used in boar diets without any adverse effects on spermatozoa quality.

1. Introduction

In animals, nutrition plays a major role in boar reproduction, including the attainment of sexual maturity, both in terms of spermatogenesis and libido. Therefore, corn, soybean meal and wheat bran are the most common ingredients used in swine diets in South Africa. They are used because of their high nutritional value and economic benefits [1]. Pig nutrition plays an important role in the regulation of the production and reproductive efficiency of boars. In South Africa, affordability of conventional feeds has gone beyond the reach of smallholder pig farmers, due to declining grain production, increasing competition with humans for feed ingredients. To sustain local production, there is a need to look for local and readily available alternatives to substitute corn, soybean meal, fish meal, and soya oil cake that is the major protein source in pig feed. This problem could be solved by introducing less expensive local feed ingredients like potato hash in pig feed formulations to reduce the overall cost of production. Availability of agro-industrial by products has enabled smallholder pig farmers to use alternative energy sources to replace cereals in pig diets [2, 3]. Potato hash (by-product produced from the processing of chips) is produced in South Africa and can be use as alternative energy sources in pig feed. Potato by-products represent an opportunity for livestock feeders because they are an inexpensive but energy-dense dietary ingredient [4]. However, less research has been devoted to study the feeding opportunities of potato hash diets on conducive reproduction of boars. A good balance between energy and protein feeds in the ration is the key factor in achieving optimum performance. It is important to evaluate the potential of liquid fermented potato hash diet on epididymal spermatozoa quality of LW × LR pigs. Feeding fermentable carbohydrates such as raw potato starch (RPS) has been shown to strongly reduce skatole concentration in the adipose tissue of barrows [5] and entire male pigs [6, 7]. As skatole breakdown in the adipose tissue occurs rapidly, when microbial formation is reduced, feeding resistant starch during the week prior to slaughter seems enough to achieve significant reductions [8]. According to reference [9], back fat thickness is thus conceivable that selection based primarily on productive characteristics, especially for lean growth, leads to reproductive problems, such as low spermatozoa production.

Spermatozoa are produced in the testis as the result of a complex assembly line that makes a highly shaped cell, morphologically and biochemically specialized. The epididymal secretome and proteome of several mammalian species include pigs. According to reference [10] one of the changes produced in the spermatozoa through its epididymal maturation was the migration of the cytoplasmic droplet from the proximal position to the distal position of the midpiece [11]. As a result, epididymal spermatozoa and often ejaculated spermatozoa contain a heterogeneous group of spermatic cells that vary in degree of maturation and show different morphologies and fertility potential. Feeding diets containing fermented liquid feed have been shown to increase pig performance and improve the microbial environment in the gastrointestinal tract [12, 13, 14], however no work was done on epididymal semen quality of LW × LR boar. Therefore, effects of liquid fermented potato hash diet on spermatozoa traits have not been examined in LW × LR boars. To the knowledge of the authors, there is no study available, to date, on the effect liquid fermented potato hash diet on spermatozoa quality of LW × LR pigs. Several factors affect spermatozoa quality in boars; photoperiod, environmental relative humidity and temperature, nutrition, handling, breed, age, viral or bacterial infections, and, especially, the frequency of semen collection significantly affect the number of spermatozoa per ejaculate as well as spermatozoa motility and morphology [15].

To date, no study has reported the effect of liquid.

2. Materials and methods

2.1 Location and experimental area

The study was conducted at Germplasm, Conservation and Reproductive Bio-technologies Unit of Agricultural Research Council (ARC), Animal Production (AP), (ARC-AP: Irene, Pretoria, South Africa). The ARC-AP campus is located at 25°55′ South; 28°12′ East. The campus is located in the Highveld region of South Africa and situated at an altitude of 1525 m above sea level. Potato hash (PH) was collected from Simba (Isando, Gauteng, South Africa), a potato chips factory in South Africa for processing and production of fermented liquid potato hash diet (FLPH).

2.2 Fermentation process and diets

A back-slopping fermentation approach was used to prepare fermented liquid potato hash diets as described by reference [16]. Fermented liquid diets were prepared by mixing potato hash diets with water, at a ratio of 1:2. The diets were formulated to provide 14 MJ/kg digestible energy (DE), 180 g crude protein (CP)/kg and 11.6 g lysine /kg which meet and exceed the requirements of growing pigs [17]. Three diets were formulated to be isoenergetic and isonitrogenous containing either 200 (LFLPH) g/kg potato hash and 400 (HFLPH) g/kg potato hash or no fermented potato hash (control). The seven dietary treatments are showed in Figure 1: A-CON (control diet non-fermented, contain no potato hash), B-LFC (liquid fermented control diet), C-LLPH (diet containing 200 g potato hash.kg⁻¹ as fed), D-HLFPH (diet containing 400 g potato hash.kg⁻¹ as fed), E-LFCE (fermented control diet treated with an exogenous xylanase enzyme (Natugrain TS L®), F-LLFPHE (diet containing 200 g potato hash.kg⁻¹ as fed treated with an exogenous xylanase enzyme (Natugrain TS L®)), G-HLFPHE (diet containing 400 g potato hash.kg⁻¹ treated with an exogenous xylanase enzyme (Natugrain TS L®). The fermented diets were stored in closed 100 L drums under agitation at 25°C for 8 hours before being fed to the pigs. Pigs were adapted to diets for a period of ten days. Both experimental diets and water were provided ad libitum for two months (Table 1) [16].

Figure 1.

Dietary treatments of liquid fermented potato hash; A- CON- control (liquid fermented control with and without enzyme); LLFPH - low inclusion of liquid fermented potato hash with and without enzyme; HLFPH - high inclusion of liquid fermented potato hash with and without enzyme; LFC + E-liquid fermented control with enzyme diet; LFC – Liquid fermented control diet.

	Control	LLFPH	HLFPH
Experimental diets1
Ingredient kg
Hominy Chop	608.7	504.4	400
Molasses	20	15	10
Potato Hash	0	200	400
Soybean Oilcake	181.4	166.7	152.1
Maize meal	150	75	0
Monocalcium Phosphate	5	8.1	11.2
Limestone	18.8	16.3	13.7
Lysine HCI	8	6.5	5
Salt	4	4	4
Vitamin-mineral Premix2	4	4	4
Calculated composition
Nutrients g/kg
DM	892	605	599
Ash	2.5	31	37
CP	180	180	180
Crude fiber	57	58	60
Calcium	9.12	9.12	9.12
Phosphorus	5.47	5.46	5.46
Lysine	11.6	11.6	11.6
Methionine	0.67	0.56	0.56
DE MJ/kg	13.5	13.5	13.5

Table 1.

Composition and chemical analysis of the diet on as-is basis of different inclusion levels of liquid fermented potato hash.

¹

CON = control (liquid fermented control with and without enzyme); LLFPH = low inclusion of liquid fermented potato hash with and without enzyme; HLFPH = high inclusion of liquid fermented potato hash with and without enzyme.

²

Provided the following per kg of diet: 6500 IU vitamin A, 1200 IU vitamin D3, 40 IU vitamin E, 2 mg vitamin K3, 1–5 mg vitamin B1, 4.5 mg vitamin B2, 0.03 mg vitamin B12, 2.5 mg vitamin B6, 25 mg niacin, 12 mg calcium pantothenate, 190.5 mg choline, 0.6 mg folic acid, 0.05 mg biotin, 40 mg manganese, 100 mg zinc, 125 mg copper, 1 mg iodine, 100 mg ferrous, and 0.3 mg selenium [16].

2.3 Characterization of the liquid fermented potato hash

Fermented liquid feed is defined as a mixture of feed and water that is stored in a tank at a specific temperature and for a specific time before being fed to animals [16]. Fermented liquid by-products, fermented diets can also be achieved when dry compound feed is mixed with water and stored for at least 8 hours [18]. This processing method can easily be used under small farmer conditions. It is also known as soaking. A short-term perseveration technique is used to store liquid by-products. Feeding fermented liquid compound feeds to weaned piglets improved daily gain and changed the gastrointestinal environment in a more desirable direction compared to non-fermented liquid feeds [14]. Feeding fermented liquid compound feeds to weaned piglets improved daily gain and changed the gastrointestinal environment in a more desirable direction compared to non-fermented liquid feeds. Processed potatoes such as hash represent a potential energy source that could replace or be included in the traditional pig diet [13]. Another disadvantage of feeding hash to growing pigs is that it contains a high-fiber diet that increases the passage rate in growing pigs [16].

2.4 Pigs, experimental design and housing

Forty-two (6 pigs per treatment) crossbred boars (Large White × Landrace) aged 55 days with an average weight of 25.5 ± 3 kg was randomly selected from the ARC-AP Irene, pig breeding unit. The boars were allocated to dietary treatments in a completely randomized design. The boars were housed individually in 1.54 × 0.8 m pens in environmentally controlled houses with the temperature ranging from 22 to 25°C. Each pen was provided with wood shaving. Daily feed offered and weekly orts were recorded. Orts were dried, weighed and discarded daily. Weights of feed refusals and orts were subtracted from the total weight of the feed allocated to determine feed intake for that week. Weight of the feed consumed each week was divided by seven to determine the average daily feed intake. Feed was supplied ad libitum and water was made available at all times through drinking nipples. Mortality and morbidities were noted. Morbidities were diagnosed and the necessary treatments were done [16].

2.5 Boars slaughtering and measurements of testicular traits

These pigs were humanely slaughtered when they attained a weight of 60 ± 4 kg. The pigs were then stunned with an electrical stunner set at 220 V and 1.8 A with a current flow for 6 s and exsanguinated within 10 s of stunning [9]. While boar was bleeding, testis and epididymis were carefully removed before the carcass was dipped into hot water, de-haired and eviscerated. The length, width and weight for left and right testis were measured. The length, width and weight for left and right testis were measured using caliper. The weight of testis was measured using a sensitive digital weighing scale. The right and left epididymis was trimmed off the body of the testis. Scrotal sacs were incised to exteriorize the testis and epididymides carefully collected, trimming off adhering tissues and weighed using a sensitive electronic balance. Other testicular and epididymal morphometric characteristics such as length, diameter, and volume of the testis, and epididymal length were also measured. The testis length, width and epididymal length were measured with the aid of a pair of Vernier calipers, while the testis volume was measured by water displacement according to Archimedes principle. Paired and mean testicular and epididymal parameters were computed from data for left and right testis and epididymis.

2.6 Semen sample collection and evaluation

The boars were slaughtered, and semen samples were collected from head of the epididymis through a razor blade incision made by on the right and left testicles. The epididymal semen samples were collected into a graduated 15 mL tube. The semen samples were placed in well-insulated flasks maintained at a warm temperature (37°C) before being transported to the laboratory within 30 minutes for measurement of semen traits categorized into microscopic evaluation.

2.7 Spermatozoa motility

Spermatozoa motility was determined using a sperm class analyzer® (CASA) (Microptic S.L, Spain). Five hundred microliters of Ham’s F-10 (Sigma-Aldrich, South Africa) and 5 μL of semen were mixed in a 1 mL graduated tube and incubated for 5 minutes at 37°C. After incubation, 10 μL of extended semen was placed on a pre-warmed microscopic slide (37°C), mounted with a cover slip and examined (×10) under a phase contrast microscope (Nikon, Japan). Spermatozoa motility was categorized as follows: Progression (%), Total motility (TM) - is a sum of progressive and non-progressive motility; Progressive motility (PM) - spermatozoa that are moving forward; Average values of velocity parameters; Curvilinear velocity (VCL) - average velocity which measures a spermatozoa movement along its actual path (μm/s); Straight-line velocity (VSL) - average velocity which measures a spermatozoa movement along a straight line from beginning to the end (μm/s); Average path velocity (VAP) - average velocity of the smoothed cell path (μm/s); Linearity (LIN) - linearity movement is a ratio of VSL/VCL (%); Straightness (STR) - straight line movement is a ratio of VSL/VAP (%) and Wobble (WOB) - wavering movement which is a ratio VAP/VCL (%) [19, 20, 21].

2.8 Spermatozoa concentration

Spermatozoa concentration was determined with a 6310 spectrophotometer (Jenway, United Kingdom). A square cuvette was filled with 3 mL of sodium citrate solution and placed in a spectrophotometer for at least 30 seconds. Raw semen (15 μL) was added in a square cuvette containing the sodium citrate solution, again placed in a spectrophotometer in order to read the absorbance. The absorbance read was used to determine the final spermatozoa concentration (10⁶/mL) with the aid of a formula (201 × 25.97 × absorbance - 0.3). The final spermatozoa concentration was recorded in millions per milliliter spermatozoa concentration. Semen pH was measured using the pH meter (Oaklon, EW35614–30, ColeParmer, East Bunker Court, Vernon Hills, IL, USA) [20].

2.9 Spermatozoa morphology

The morphology was determined microscopically after staining the semen samples with Eosin Nigrosin stain (Onderstepoort, Pretoria) on a slide. Boar semen was added to 20 μL Eosin Nigrosin staining solution in a 0.6 mL micro-centrifuge graduated tube and mixed gently. A drop of 5 μL boar semen and Eosin Nigrosin stain was placed on a clear end of a microscope slide and smeared. Semen samples was determined using Eosin Nigrosin stain (pH - 8.39), to determine percentage live or dead spermatozoa and evaluation of the spermatozoa morphology (normal or abnormal). The spermatozoa smears were prepared on a clean, warm microscope slide to avoid temperature shock to the spermatozoa and evaluated on the same day of semen collection and with the aid of a fluorescent microscope (BX 51TF) using an oil immersion objective (×100 magnification). Live spermatozoa were further evaluated for spermatozoa morphology and abnormalities. Abnormalities of the spermatozoa were categorized as primary (small, large or swollen head, double heads, abnormal acrosome, elongated and mid-piece, double and short tail), secondary (detached, loose or damaged acrosomes, bent and protoplasmic droplets of the mid-piece, bent and shoe-hook tail) and tertiary abnormalities (reacted acrosomes and coiled tails), such as live, dead, distal droplet, head, midpiece and tail [21].

2.10 Membrane integrity

Membrane integrity (Figure 2) was assessed using the osmotic resistance test (the hypoosmotic swelling test – HOST) by incubating an aliquot (100 μL) of semen sample with 1 ml of double distilled water at 37°C for 30 minutes [22]. After incubation, a pinch of Eosin was added; a drop of the well-mixed sample was placed on a glass slide and covered with cover slip. This slide was observed at 400× magnification under the phase contrast microscope. Spermatozoa with swollen tail were counted as HOST positive. A minimum of 200 spermatozoa were observed for tail coiling (Figure 2). The percentage of reactive spermatozoa was then calculated by subtracting the percentage of tail defects recorded in the sperm population before incubation in HOST media was carried out.

Figure 2.

Membrane integrity of the raw boar spermatozoa evaluated with HOST. (A) Spermatozoa with intact membrane and (B) Spermatozoa with damaged membrane.

2.11 Statistical analysis

The liquid fermented potato hash and genotype on epididymis spermatozoa quality, testicular development was performed using (SAS) version 9.3 statistical software (SAS, 1999). The GLM procedure was also used to determine the effect of LLFPH, HLFPH, LFC + E, LFC, LLFPH+E, HLFPH+E and genotype. A 5% significance level was used.

3. Results

3.1 Epididymal semen volume, semen pH, spermatozoa concentration, and abnormalities spermatozoa morphology

The effect of supplementation liquid fermented potato hash diet on epididymal semen volume, spermatozoa concentration, semen pH and abnormalities spermatozoa morphology, in LW × LR boars are shown in Figure 3. However, LFC and HLFPH+E had lower (P < 0.05) epididymal semen volume compared to control, LLFPH, HLFPH, LFC + E and LLFPH+E treatments. There was no difference (P > 0.05) in epididymal semen pH between the treatments. There was a difference (P < 0.05) in epididymal spermatozoa concentration between the treatments. Boars fed HLFPH+E had lower spermatozoa concentration compared to boars consuming control, LLFPH, HLFPH, LLFPH+E, LFC + E and LFC treatments. There was a difference (P < 0.05) in epididymal head abnormalities spermatozoa between the treatments. Where boars fed LLFPH had higher head abnormalities spermatozoa compared to control, HLFPH, LLFPH+E, HLFPH+E, LFC + E and LFC treatments. There was a difference (P > 0.05) in epididymal tail abnormalities spermatozoa between the different treatments. Boars fed control treatment had lower epididymal tail abnormalities spermatozoa compared to boars on the other dietary treatments.

Figure 3.

The effect of supplementation liquid fermented potato hash diet on epididymal semen volume, spermatozoa concentration, semen pH and abnormalities spermatozoa morphology, in LW × LR boar (±SEM). ^abcValues with different superscripts within a row differ significantly (P < 0.05, SEM- standard error of mean, SC – Sperm concentration; CON- control (liquid fermented control with and without enzyme); LLFPH - low inclusion of liquid fermented potato hash with and without enzyme; HLFPH - high inclusion of liquid fermented potato hash with and without enzyme; LFC + E-liquid fermented control with enzyme diet; LFC - Liquid fermented control diet.

3.2 The effect of supplementation liquid fermented potato hash diet on epididymal spermatozoa parameters

The mean supplementation liquid fermented potato hash diet on epididymal spermatozoa motility and velocity parameters of LW × LR boar semen as measured by CASA are shown in Table 2. There was a difference (P < 0.05) in total and progressive spermatozoa motility between the treatments. Boars fed LLFPH had higher epididymal total motility spermatozoa and progressive motility compared to boars on all the other dietary treatments. Boars fed LFC had epididymal lower progressive motility compared to the control, LLFPH, HLFPH, HLFPH+E, LLFPH+E and LFC + E treatments. There was a difference (P < 0.05) in both rapid and slow epididymal spermatozoa between the treatments., Boars fed HLFPH+E had lower epididymal rapid spermatozoa compared to control, LLFPH, HLFPH, LLFPH+E, LFC + E and LFC treatments. There was an increase in slow spermatozoa with the increases of HLFPH+E treatments. There was no difference (P > 0.05) in VCL spermatozoa between the treatments. However, pigs fed HLFPH+E tended to have decreased VCL spermatozoa. There was a difference (P < 0.05) in epididymal VSL spermatozoa between the treatments. However, pigs fed control (35.08) had higher values VSL epididymal spermatozoa compared to LLFPH, HLFPH, HLFPH+E, LLFPH+E, LFC + E and LFC (27.93, 24.53, 26.55, 32.15, 25.70 & 23.62) treatments. There was no difference (P > 0.05) in epididymal VAP spermatozoa between control, LLFPH, HLFPH, LLFPH+E, LFC + E and LFC but there was difference (P < 0.05) with HLFPH+E treatments. There was no difference (P > 0.05) in epididymal linearity between the treatments. However, pigs fed LLFPH+E had lower epididymal linearity spermatozoa compared to control, LLFPH, HLFPH, HLFPH+E, LFC + E and LFC treatments. There was no difference (P > 0.05) in epididymal wobble spermatozoa between the control, HLFPH, HLFPH+E, LLFPH+E, LFC + E and LFC treatments, however there were difference with LLFPH treatment. There was no difference (P > 0.05) in ALH epididymal spermatozoa between the treatments. There was no difference (P > 0.05) in BCF spermatozoa between the treatments. However, pigs fed LFC + E had higher BCF spermatozoa compared to control, LLFPH, HLFPH, LLFPH+E, HLFPH+E, and LFC treatments. Pigs fed control diet had higher medium spermatozoa compared to the LFC, LLFPH, HLFPH, HLFPH+E, LLFPH+E and LFC + E treatments. There was a difference (P < 0.05) in straightness spermatozoa between the treatments. However, pigs fed LFC had higher epididymal straightness spermatozoa compared to the control, LLFPH, HLFPH, HLFPH+E, LLFPH+E and LFC + E treatments. There was a difference (P < 0.05) in statics spermatozoa between the treatments. However, pigs fed HLFPH+E had an increase in epididymal statics spermatozoa compared to the control, LFC, LLFPH, HLFPH, LLFPH+E and LFC + E treatments.

Parameters	Control (n = 6)	LLFPH (n = 6)	HLFPH (n = 6)	LFC + E (n = 6)	LFC (n = 6)	LLFPH + E (n = 6)	HLFPH + E (n = 6)	SEM	P-value
	TM (%)	80.68^a	83.68^a	78.47^a	69.02^bc	64.73^c	74.03^b	78.58^a	1.989	<.0001
PM (%)	64.40^b	70.93^a	69.62^a	57.45^bc	40.07^c	57.02^bc	63.67^b	3.391	<.0001
RAP (%)	72.37^a	58.53^ab	47.62^bc	52.15^b	57.45^b	60.70^ab	49.43^c	5.055	0.0271
SLW (%)	2.67^c	3.48^c	4.68^bc	5.52^ab	4.72^bc	3.10^c	7.03^a	0.639	0.0003
VCL (μm/s)	138.00^a	131.75^a	140.55^a	134.24^a	113.93^a	129.09^a	114.63^a	11.470	0.5301
VSL (μm/s)	35.08^a	27.93^ab	24.53^b	26.55^bc	32.15^a	25.70^bc	23.62^c	2.308	0.0108
VAP (μm/s)	65.98^a	57.80^a	56.47^a	57.12^a	58.75^a	59.38^a	45.92^b	4.401	0.1221
LIN (%)	25.78^a	22.13^a	22.75^a	19.75^a	25.25^a	20.08^a	26.92^a	2.808	0.4367
WOB (%)	46.30^a	37.10^b	42.77^a	42.43^a	46.30^a	43.15^a	43.17^a	2.660	0.2646
ALH (%)	4.47^a	4.47^a	3.93^a	3.90^a	4.40^a	3.77^a	4.27^a	0.275	0.2993
BCF Hz	8.40^b	7.17^b	9.08^b	12.67^a	11.00^a	8.95^b	8.17^b	1.109	0.0229
MED (%)	20.45^b	19.68^b	31.00^a	15.70^bc	11.60^bc	12.37^bc	5.72^c	2.289	<.0001
STR (%)	49.08^a	49.58^a	46.88^b	46.67^b	54.75^a	45.22^b	53.58^a	1.969	0.0106
Static (%)	5.167^c	18.15^bc	24.30^b	26.63^b	26.23^b	23.83^b	37.82^a	4.459	0.0010
Live spermatozoa (%)	81.03^a	80.68^a	80.80^a	52.83^c	59.83^bc	70.67^b	64.50^b	1.679	<.0001
Dead spermatozoa (%)	10.02^b	7.78^b	7.37^b	15.17 ^a	6.00^b	3.83^c	3.67 ^c	0.915	<.0001
HOST (%)	76.98^a	76.43^a	80.00^a	75.42^a	80.30^a	64.50^b	65.65^b	2.911	0.0009

Table 2.

Influence of liquid fermented potato hash inclusion on semen and spermatozoa characteristics of LW × LR boars (+SEM).

^abcValues with different superscripts within a row differ significantly (P < 0.05). TM - Total motility, PM - Progressive motility, SLW - Slow, MED - Medium. RAP - Rapid, VCL - Velocity curvilinear, VSL - Velocity straight line, VAP - Velocity average pathway, LIN - Linearity, STR - Straightness, WOB - Wobble, ALH - Amplitude, BCF- Frequency, CON - control (liquid fermented control with and without enzyme), LLFPH - low inclusion of liquid fermented potato hash with and without enzyme, HLFPH - high inclusion of liquid fermented potato hash with and without enzyme and HOST - hypo-osmotic swelling test.

3.3 The effect of supplementation liquid fermented potato hash diet on epididymal morphology and membrane integrity parameters

Analysis of boar spermatozoa morphology with Eosin/Nigrosin staining solution viewed under a fluorescence microscope at 100x magnification is indicated in Figure 4. Boars fed LFC + E had lower live spermatozoa compared to control, LLFPH, HLFPH, LLFPH+E, HLFPH+E and LFC treatments. There was a difference (P < 0.05) in dead epididymal spermatozoa between the treatments. Although pigs fed LFC + E had higher dead epididymal spermatozoa compared to the control, LFC, LLFPH, LLFPH+E, HLFPH and LLFPH+E treatments. Membrane integrity of the raw boar spermatozoa evaluated with HOST shown in Figure 3. The HLFPH and LFC had a higher (P < 0.05) osmotic swelling test compared to control, LLFPH, LLFPH+E, LFC + E and LLFPH+E treatments.

Figure 4.

Analysis of boar spermatozoa morphology with eosin/Nigrosin staining solution viewed under a fluorescence microscope at 100x magnification. (A): Live spermatozoa and (B): Dead spermatozoa.

3.4 Testicular and epididymal morphometric

Testicular and epididymal morphometric characteristics of White × Landrace boars fed liquid fermented potato hash diets are show in Table 3. There was a difference (P < 0.05) in the right testis weight and width indexes between the treatments. Boars fed HLFPH+E had higher right testis weight index compared to the control, LLFPH, HLFPH, LFC + E, LLFPH+E and HLFPH+E treatments. Pigs fed HLFPH had lower right testis width index compared to the control, LLFPH, LFC + E, LFC, LLFPH+E, and HLFPH+E treatments. The LLFPH+E and LLFPH had a higher (P < 0.05) left testis weight index compared to control, HLFPH, LFC + E, LFC, and HLFPH+E treatments. Boars fed LFC + E had higher left testis width index compared to the control, LLFPH, HLFPH, LFC, LLFPH+E and HLFPH+E treatments. However, there was no difference (P > 0.05) in left testis width and length index between the treatments. Boars fed HLFPH had the lowest right testis length index compared to all the other treatments. There was a difference (P < 0.05) in right epididymis length between the treatments, with boars consuming HLFPH and LLFPH diets having the shortest right epididymis length. There was a difference (P < 0.05) in left epididymis length between the dietary treatments, demonstrated by the shortest left epididymis in boars that were fed HLFPH and LLFPH+E diets.

Variables	Control (n = 6)	LLFPH (n = 6)	HLFPH (n = 6)	LFC + E (n = 6)	LFC (n = 6)	LLFPH + E (n = 6)	HLFPH + E(n = 6)	SEM	P-value
	Right testis weight index (kg)	0.96^a	0.86^ab	0.74^b	0.99^a	0.88^ab	0.88^ab	1.00^a	0.032	<.0001
Right testis width index mm	2.07^a	1.81^ab	1.50^b	2.22^a	2.00^a	1.81^ab	2.20^a	0.098	<.0001
Left testis weight index (kg)	0.95^a	0.85^ab	0.73^b	0.98^a	0.86^ab	0.87^b	0.98^a	0.031	<.0001
Left testis width index (mm)	2.00^b	1.77^bc	1.46^c	3.03^a	2.00^b	1.78^bc	2.15^b	0.350	0.0900
Right testis length index (mm)	0.97^a	0.86^bc	0.73^c	0.96^a	1.00^a	0.90^a	0.97^a	0.026	<.0001
Left testis length index, (mm)	0.98^a	0.85^ab	0.74^b	0.95^a	0.98^a	0.88^ab	0.96^a	0.025	<.0001
Right epididymis length (mm)	0.53^a	0.47^a	0.37^b	0.53^a	0.50^a	0.44^b	0.49^a	0.018	<.0001
Right epididymis weight index (g/kg)	3.21^a	2.94^a	3.26^a	2.91^a	2.87^a	2.20^b	2.46^ab	0.268	0.0830
Left epididymis length (mm)	0.53^a	0.46^ab	0.36^b	0.51^a	0.49^a	0.44^b	0.48^a	0.018	<.0001
Left epididymis weight index (mm)	2.91^a	2.75^a	3.04^a	2.88^a	2.87^a	2.21^b	2.46^ab	0.241	0.2138

Table 3.

Influence of dietary liquid fermented potato hash inclusion on testicular development for white × landrace boars (+SEM).

^abMeans on same row with different superscripts differ significantly (P < 0.05), SEM - standard error of mean, C - control (liquid fermented control with and without enzyme); LLFPH - low inclusion of liquid fermented potato hash with and without enzyme; HLFPH - high inclusion of liquid fermented potato hash with and without enzyme; LFC + E - Liquid fermented control with enzyme diet; LFC - Liquid fermented control diet.

4. Discussion

4.1 The effect of supplementation liquid fermented potato hash diet on semen volume, pH, spermatozoa concentration and sperm motility parameters

The study hypothesized that epididymal spermatozoa quality was affected by inclusion levels of liquid fermented potato hash supplementation in LW × LR boars. Supplementation of liquid fermented potato hash in our study did not show any significant negative effect on semen volume of LW × LR pigs. Similarly, Ogunlade et al. [1] reported that dietary supplementation with Saccharomyces cerevisiae to layer breeders improved semen quality by increasing semen volume, sperm concentration, and motility and by reducing dead and abnormal sperm as opposed to the untreated group. There were no significant differences in the epididymal semen volume were recorded in this study. Similarly, Santos et al. [23] who observed no effect of palm kernel cake (PKC) and coconut meal (CM) diets on semen volume, gross motility, vigor, and spermatozoa morphologic defects of the ejaculates in water buffalo semen. Wähner et al. [24] also reported an increase in semen volume and higher spermatozoa concentration, as l-carnitine increased in boars. In our study the inclusion levels of liquid fermented potato hash did not show any significant effect on spermatozoa concentration [25]. Similar results were obtained with male buffalo calves, in which the supplementation of yeast fermentation product (0%,0.5%, or 1.0%) of the diet did not impact sperm concentration, volume, motility, and viability [18]. A significant increase was recorded in the epididymal sperm concentration and motility of rats consuming fermented rooibos and ‘green’ rooibos when compared with the control group and other experimental groups [26]. No significant difference in the epididymal head abnormalities spermatozoa was observed in contrast to the findings of [2, 3] that no effect of (200, 400 and 600 mg/kg of sweet potato (Ipomoea batatas) leaf extract as compared to the control diet on spermatozoa concentration on semen in rabbit.

Amao and Showunmi [27] reported that rabbit increases epididymal spermatozoa concentration in bucks fed control and fermented cottonseed cake than bucks fed raw cottonseed cake-based diet. Similarly, Chung et al. [28] found that Lepidium meyenii (Maca) aqueous extract increased the epididymal sperm count of a rat. Ekpo et al. [29] reported that the recent evidence indicates that there was no change in epididymal spermatozoa parameters from the rats fed sweet potato. In the present study, semen pH was not affected by liquid fermented potato hash supplementation in Large White x Landrace pigs was expected. Ekpo et al. [29] reported that increases of epididymal semen pH, as inclusion levels of sweet potato increased in albino rats. The semen pH of the ejaculates ranged between 6.8 and 7.0 with no difference among the palm kernel cake (PKC) and coconut meal (CM), also in accordance to previous findings Sansone et al. [30, 31]. According to Uno et al. [32] there was no significant difference in the epididymal semen pH among the different treatment groups. The pH of the semen was between the range of 7.18 to 7.25 in the current study as shown in Table 2. Uno et al. [2, 3] reported a significant decrease in the weight of epididymal spermatozoa count in albino rats fed/supplemented with the leaves extract. Amao and Showunmi [27] found that spermatozoa count increased with fermented cottonseed cake diet. While Etchu et al. [33] found that semen volume, spermatozoa concentration and sperm output decreased with processed sweet potato in diet. An increase in total motility spermatozoa suggests that including liquid fermented potato hash beyond (diet containing 200 g potato hash.kg⁻¹ diet) compromises motility of spermatozoa. An increase in total motility spermatozoa found in the current study correspond with finds reported by Amao and Showunmi [27] of the highest spermatozoa motility in rabbit supplemented with 200 mg fermented cottonseed cake. Pant et al. [34] and dos Santos et al. [23] indicated that progressive spermatozoa motility was higher in water buffalo fed with a diet containing 69.3% palm kernel cake. In addition, Rubus coreanus has been reported to increase spermatozoa counts and motility in white rabbit [10]. The increase in sperm output by testis size line boars was due to larger testis and greater rates of daily sperm production [35]. The observation that progressive motility was affected by inclusion levels of liquid fermented potato hash in LW × LR boars. Dos Santos et al. [23] reported that progressive spermatozoa motility of buffalo with supplemented palm kernel cake was increased than the rabbit fed control group. Zhao et al. [36] reported that, these results suggested that wine grape pomace could be used as a feed ingredient in rams to alleviate restraint induced oxidative stress and improve epididymal spermatozoa quality. Etchu et al. [33] reported that semen volume, spermatozoa concentration (109/cm³), spermatozoa motility (%), live/dead ratio (%), spermatozoa output (109/cm³), abnormal spermatozoa (%), semen pH, birds fed fermented sweet potato-based diets was decreases than the birds fed control diet, sliced potatoes, and grated potatoes. McDaniel et al. [37] reported that the effect of yeast fermentation product on the semen quality index was mathematically due to a reduction in spermatozoa motility in White Leghorn roosters. In White Leghorn roosters, [38] cited that that spermatozoa motility of White Leghorn roosters supplemented with fermentation product to roosters linearly decreased possibly due to the linear increase in the number of bacteria per spermatozoa and yeast per spermatozoa. Uno et al. [2, 3] who observed significant effect of on the weight of epididymes, spermatozoa motility and spermatozoa viability in albino rats treated the leaves extract. Similarly, van Dorland et al. [39] suggested that the supplementation of yeast product can improve semen quality in horses by increasing the antioxidant capacity in the semen. However, mammals and birds exhibit remarkable differences in their reproductive systems, so it is possible that the yeast benefits reported in rats would not apply to avian species. Abaza et al. [40] reported that dietary supplementation with Saccharomyces cerevisiae to layer breeders improved semen quality by increasing semen volume, sperm concentration, and motility and by reducing dead and abnormal sperm as opposed to the untreated group.

4.2 The effect of supplementation liquid fermented potato hash diet on spermatozoa abnormalities

An increase in live spermatozoa suggests that including potato hash beyond control diet (not fermented and without potato hash) compromises livability of spermatozoa. An increase in live spermatozoa found in the current study correspond well with finds by Ragab et al. [41] who reported a highest total motile, total live and total normal sperm in rabbits supplemented with 2.5 g pumpkin seed oil/kg diet plus 2.5 g black seed oil/kg diet /kg diet (PSO + BSO). Ekpo et al. [29, 42] indicated that number of abnormal and head spermatozoa was lower in albino rats fed with a diet containing 200 mg/kg of sweet potato. Findings of Bréque et al. [43] does not support the outcomes of the present study that increasing levels of liquid fermented potato hash decreased dead spermatozoa and head abnormalities. A study by Lovercamp et al. [19] regarding farrowing rate revealed that boars with a low fertilization performance had a significantly lower pro-portion of normal spermatozoa than boars with a high performance. Njoku et al. [44] highlighted that an addition of pumpkin seed reduced considerably the percentage of morphologically changed spermatozoa while the sperm count, motile sperm and viability improved. Skoracka et al. [45] reported that high levels of dietary zinc supplementation preservation sperm morphology, sperm count and function, and thus, for the proper course of fertilization. It worth noting that liquid fermented potato hash requirement for pigs, although some of these gave good results, it is difficult to obtain a reliable and consistent fermentation due to some factors.

Supplementation of liquid fermented potato hash diet in our study did not show any significant effect on live and dead spermatozoa. No significant difference in the spermatozoa live and head abnormalities was similar to the findings of Ekpo et al. [29] who observed no effect of inclusion levels of sweet potato (Ipomoea batatas) on spermatozoa live and head abnormalities in male albino rats. Etchu et al. [33] reported that there was a decrease of live/dead ratio (%), abnormal spermatozoa (%) of birds fed fermented sweet potato-based diets than birds fed control diet, sliced potatoes, and grated potatoes. Gofura et al. [20] reported that the abnormality of spermatozoa increased with dietary of purple sweet potato increased in rats. Morphologically normal spermatozoa (50.67%) were also significantly lower than that of the fermented cottonseed cake group but similar to that of the control [27]. However, non-motile spermatozoa, abnormal spermatozoa, round and elongated spermatids were not significantly affected by fermented cottonseed cake diets [27]. Dietary supplementation of yeast fermentation product leads to a linear decrease in the spermatozoa quality index, which is indicative of overall semen quality and is affected by spermatozoa viability, concentration, and motility [37]. The results obtained by Uno et al. [2, 3] revealed a significant decrease in the spermatozoa viability and spermatozoa count while spermatozoa head abnormalities significantly increased in albino rats treated the leaves extract.

4.3 The effect of supplementation liquid fermented potato hash diet on testicular, epididymal weight and size

An increase in testicular weight as supplementary PH levels increased is similar to findings of Ekpo et al. [29] higher testis weights and epididymes testis length and width in rats supplemented 600 mg/kg of sweet potato (Ipomoea batatas) than others group and control diet. In contrast, increasing levels of leaf meal inclusion resulted in decreased testicular size of rabbits [46] Testicular weights have been reported to have a high correlation with sperm reserve in the testis or epididymis and therefore a reflection of sperm production [46]. In rabbits, Ansa et al. [47] find that testis length, testis circumference, testis weight, testis volume, epididymis weight of rabbit supplemented with 900 mg kg⁻¹ methanolic extract of Phoenix dactylifera fruit per day was bigger than the un-supplemented group. Peerry and Petterson [48] reported that size, length, and width of testis are good indicators of present and future sperm production. In the present study, weight epididymis was affected by dietary levels of liquid fermented potato hash supplementation in LW × LR pigs. An increase on right testis weight index as supplementary levels HLFPH+E increased is similar to findings of Adienbo and Wodu [49] where sweet potato supplementation increased weights of the testis and epididymes in animals. In contrast Uno et al. [2, 3] reported that testis weight was not affected by sweet potato (Ipomoea batatas) 200, 400 and 600 mg/kg when compared with the control diet in rats. Amao and Showunmi [27] reported that bucks fed on raw cottonseed cake -based diet had significantly higher values for left, right and mean epididymal weights than for bucks on other fermented cottonseed cake. Epididymis stores and transports spermatozoa that are produced in the testis of boars [50].

An increase in epididymis weight was expected. Testicular testosterone output is primarily regulated by the pulsatile pattern of pituitary LH secretion [51]. These androgenic properties enhance concentration of luteinizing hormone, which is responsible for testicular development such as epididymis and testicular weight [50]. An increase in epididymis weight could be is associated with quadratic increase in semen volume. Amao and Showunmi [27] reported increased epididymides (left, right and mean) when rabbit bucks were fed fermented cottonseed cake compared to those fed control diet. In addition, Hyacinth et al. [21] reported a decrease on the morphometric, weights of testis and epididymides with lower inclusion of tephrosia bracteolate leaf meal. Bitto et al. [52] reported that a decreased on paired of testis weight of cockerels fed supplementation of cassava peel meal up to 30% in diet. Majid et al. [53] reported an increased size, weight, and relative weight of testis and epididymis of rats administered with 300 mg/kg day of sweet potato.

5. Conclusions

From the results of the present study the following main conclusions can be drawn:

1. liquid fermented low potato hash diet could be an alternative feed source for growing boars.

2. Therefore, the results indicated that diet contain LFLPH can be used in growing boars without any adverse effects on semen quality of boars.

3. It is recommended that further investigations on fed fermented liquid potato hash on semen collected from living boars should evaluated for semen quality, testicular morphology, and histology.

4. The findings showed that LFLPH-containing feeds may be utilized in pig diets without having any negative effects on the quality of spermatozoa.

Acknowledgments

The authors would like to express gratitude to the Agricultural Research Council (ARC) - Nutrition and Germplasm Conservation and Reproductive Biotechnologies (GCRB) staff for the assistance and Gauteng Department of Agriculture and Rural Development (GDARD). Dedication goes to the late Dr. R.S Thomas who assisted with providing funding from GDARD), and the staff at the Agricultural Research Council-Irene who assisted with the management of pigs throughout the trial are also acknowledged.

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

The experimental procedures used in this study were in accordance with guidelines of the Agricultural Research Council Animal Ethics committee (Reference: APIEC16/037), during the period from November 2016 to August 2017.

Abbreviations

ALH	amplitude of lateral head displacement, μm
ARC	Agricultural Research Council
AP	Animal production
BCF	beat-cross frequency, Hz
BSO	black seed oil
CASA	Computer-Assisted Sperm Analysis
CON	control diet
CP	crude protein
CM	coconut meal
DE	digestible energy
SC	sperm concertation, 106/mL
GCRB	Germplasm Conservation and Reproductive Biotechnologies
GDARD	Gauteng Department of Agriculture and Rural Development
LLFPH	low inclusion of liquid fermented potato hash with and without enzyme, g/kg
HLFPH	high inclusion of liquid fermented potato hash with and without enzyme, g/kg
HOST	hypo-osmotic swelling test
HLFPH+E	high liquid fermented potato hash with enzyme diet, g/kg
LLFPH+E	low liquid fermented potato hash with enzyme diet, g/kg
LFC + E	Liquid fermented control with enzyme diet, g/kg
LFC	liquid fermented control diet, g/kg
LW × LR	Large White × Landrace
MED	medium, %
NPM	non progressive motility, %
SC	spermatozoa concentration, 106/mL
PSO	pomegranate seed oil
VSL	straight-line velocity, μm/s
STR	straightness, %
LIN	linearity, %
RPS	raw potato starch
LH	luteinizing Hormone
WOB	Wobble, %
TM	total motility, %
VAP	average path velocity, μm/s
PM	progressive motility, %
μL	microliter
VCL	curvilinear velocity, μm/s
%	percentages
PKC	palm kernel cake
°C	degree celscius
SLW	slow, %
RAP	rapid motility, %
STC	static, %