Mimicking the cauda epididymal plasma-like osmolality in extender improves liquid preservation of ram semen at 3–5°C

ABSTRACT

Cauda epididymis in mammals is known to store mature sperm largely in quiescent state for several weeks without significantly affecting fertility. Hence, the aim of this study was to evaluate the effects of mimicking cauda epididymal plasma (CEP)-like conditions in extender on liquid preservation of ram semen at 3–5°C. Four experiments were conducted in this study: (1) evaluation of physicochemical properties of ram CEP, (2) effect of hyperosmotic solution on sperm motility and functional membrane integrity (FMI), and the effects of (3) CEP-like hyperosmolality (390 vs. 360 mOsmol/kg) and (4) pH in extender (pH 6.5 vs. 6.8) on liquid preservation of ram semen. Sperm treatment with hyperosmotic solution (450 mOsmol/kg) resulted in a decline (P < 0.05) in mass motility (3.5 ± 0.2 vs. 4.3 ± 0.2) and FMI (30.4 ± 3.2 vs. 52.1 ± 2.1%) compared to that with isoosmotic solution (360 mOsmol/kg). Overall, sperm viability, acrosomal integrity, and progressive motility were similar (P > 0.05) while straight-line velocity (77.8 ± 3.1 vs. 71.3 ± 2.7µm/s), linearity (47.4 ± 0.4 vs. 39.5 ± 0.9%), straightness (79.7 ± 0.5 vs. 74.0 ± 0.5%) and beat cross frequency (28.6 ± 0.8 vs. 26.0 ± 0.5 Hz) were higher (P < 0.05) and FMI (65.7 ± 1.5 vs. 75.4 ± 1.1%) was lower (P < 0.05) following liquid-preservation in hyperosmotic extender compared to that in isoosmotic extender. Both total motility (83.3 ± 1.8 vs. 75.4 ± 1.5%) and progressive motility (51.7 ± 2.3 vs. 39.5 ± 1.9%) were higher (P < 0.05) at 48 h of storage in hyperosmotic extender compared to the control. Overall, the seminal attributes were similar (P > 0.05) between the two pH’s of the extender. In conclusion, semen extender having CEP-like osmolality but not the pH was superior to extenders having conventional osmolality and pH for liquid preservation of ram semen.

Abbreviations: AI: artificial insemination; ALH: amplitude of lateral head displacement; BCF: beat cross frequency; CASA: computer-assisted semen analyzer; CEP: cauda epididymal plasma; ELON: elongation; EYTF: egg yolk-Tris-citrate-fructose; FMI: functional membrane integrity; GLM: general linear model; GPC: glycerophosphatidylcholine; HOS: hypoosmotic swelling; LIN: linearity; pHe: external pH; PROG: progressive motility; S.E.M.: standard error of the mean; SLTF: soya lecithin-Tris-fructose extender; SP: seminal plasma; STR: straightness; VAP: average path velocity; VCL: curvilinear velocity; VSL: straight-line velocity; TM: total motility

KEYWORDS:

• Ram
• semen
• liquid preservation
• cauda epididymal plasma-like pH
• osmolality

Introduction

Artificial insemination (AI) in sheep with cryopreserved semen has not been adopted widely mainly due to very poor conception rate following cervical AI (Byrne et al. 2000; Salamon and Maxwell 2000; Donovan et al. 2004). In contrast, cervical AI in sheep with fresh diluted semen yields much higher conception rate (Maxwell and Salamon 1993; Pervage et al. 2009; De et al. 2016). However, the very short shelf-life (10 to 14 h) of chilled semen has restricted its application at distant places (Lopez-Saez et al. 2000). This decline in fertility of liquid preserved semen with time is irrespective of diluents, temperature and other conditions (Paulenz et al. 2002; Pervage et al. 2009; O’Hara et al. 2010) and considered to be attributed by peroxidative damage in the sperm ultrastructures including plasma membrane (Paul et al. 2017).

The cauda epididymis in mammalian species is known to store mature sperm in a quiescent state for several weeks without significantly affecting their fertility. Therefore, sperm might be effectively preserved in vitro in an environment that closely mimics the cauda epididymal conditions by favoring sperm quiescence. The luminal fluid of distal cauda epididymis in mammals has an acidic pH (pH 6.3–6.5; Paul et al. 2018a), high osmolality (365–395 mOsmol/kg; Tamayo-Canul et al. 2011; Paul et al. 2018a) and contains high levels of carnitine (15–19 mM), glycerophosphatidylcholine (GPC, 21–97 mM; Jones 1978), total lipids, phospholipids, sialic acid, and total protein (Turner 1979), while negligible amount of utilizable energy (glucose and fructose; Verma and Chinoy 2001). Ram cauda epididymal plasma (CEP) like other mammals was reported to contain high concentration of protein (Paul et al. 2018a), K⁺ (24–39 mM) and low levels of Na⁺ (22–57 mM) and Ca²⁺ (0.48–0.95 mM) (Jones 1978; Brooks 1990; Turner et al. 1995). The specific role of these CEP factors on sperm quiescence has been extensively studied in rodents (Turner and Giles 1982; Verma and Chinoy 1985) and bovine (Carr and Acott 1984); however, similar investigations in sheep are relatively few (Gatti et al. 1993). In rodents, NaCl is essential for the motility of cauda epididymal sperm; however, the role of KCl or CaCl₂ is secondary for sperm motility (Verma and Chinoy 2001). The investigations pertaining to the effects of CEP factors on liquid preservation of ram semen are relatively few. A previous study by De Pauw et al. (2003) revealed that ram cauda epididymal sperm when diluted in Hepes-TALP media and stored at ambient temperature were preserved better at pH 6.0 and osmolality 300 mOsmol/kg compared to other pH and osmolalities. Tamayo-Canul et al. (2011) reported that the motility of ram cauda epididymal sperm was higher in TES-Tris-fructose extender having osmolality 320 mOsmol/kg than the extender having osmolality 370 or 420 mOsmol/kg following liquid preservation at 5°C. However, the acrosomal damage was higher with time in the extender having osmolality 320 or 370 mOsmol/kg as compared to the extender having osmolality 420 mOsmol/kg. In another study, a synthetic extender having ionic composition similar to that of CEP and containing both egg yolk and glycerol was found to preserve the motility of bovine sperm better than the Tris-fructose extender following liquid preservation (Verberckmoes et al. 2004). Considering the crucial role of osmolality on cellular metabolism, in general, and proven influence of osmolality of extender on semen preservation together with the characteristic higher osmolality and prolong sperm storage in CEP, it could be hypothesized that sperm functions would be preserved better following liquid preservation in an extender that has osmolality similar to CEP.

In view of the above, the current study was carried out to examine (1) the physicochemical properties of ram CEP, and to evaluate the effects of (2) short-term incubation in hyperosmotic solution and (3) liquid preservation in extenders having CEP-like osmolality or pH on various sperm quality attributes of ram semen.

Results and discussion

Physicochemical properties of ram cauda epididymal plasma

The mean values of pH, osmolality, and concentration of Na⁺ and K⁺ in ram CEP are presented in Table 1. The results showed that the values obtained in the present study were similar to those reported previously (Jones 1978; Tamayo-Canul et al. 2011).

Table 1. Physicochemical properties of ram cauda epididymal plasma.

Biophysical property	Mean	Range	Previous report^#
pH	6.32 ± 0.01	6.2–6.4	6.2–6.7^a
Osmolality (mOsmol/kg)	375.45 ± 6.44	336–421	365–395^a
Na^+ (mmol)	34.73 ± 1.06	28–45	40 (22–57)^b
K^+ (mmol)	29.15 ± 0.74	26–36	29 (24–39)^b

Data represents mean ± S.E.M. of 20 samples.

^# Data was compiled from the following sources:

^aTamayo-Canul et al. (2011)

^bJones (1978)

The mass motility of ram sperm was lower (P < 0.05) following treatment with one of the hyperosmotic solutions (i.e., 450 mOsmol/kg) as compared to the isoosmotic control (360 mOsmol/kg; Table 2). The functional membrane integrity (FMI) as determined by hypoosmotic swelling (HOS) response was diminished (P < 0.05) following pretreatment of sperm with both of the hyperosmotic solutions (390 and 450 mOsmol/kg) compared to the control (360 mOsmol/kg). However, sperm viability following the HOS assay was similar (P > 0.05) among the treatment groups and control. The decline in sperm motility in hyper-osmotic solution was probably due to osmotic extrusion of water from sperm which increased the intracellular ionic concentration and affected the cellular metabolism. In addition, the diminished HOS response following pre-exposure of sperm to the hyperosmotic solution (450 mOsmol/kg) apparently indicated loss of functional integrity of the plasma membrane. However, the absence of any significant change in the sperm viability following the incubation in hyperosmotic solution (450 mOsmol/kg) suggested that the sperm membrane may not be compromised in this solution; rather the decline in HOS response might be due to osmotic shock resulting from the higher (300 mOsmol/kg) osmotic difference between the HOS buffer and hyperosmotic solution compared to that between the HOS buffer and control (210 mOsmol/kg). Thus, the above results together suggested that treatment of ram sperm in hyperosmotic solution does not affect sperm viability; however, diminishes sperm motility and HOS response.

Table 2. Effect of pre-incubation in hyperosmotic solution on mass motility, hypoosmotic swelling response and sperm viability after the hypoosmotic swelling test.

Sperm attributes	Sodium citrate-fructose solution (mOsmol/kg)
	360	390	450
Mass motility (0–5 scale)	4.3 ± 0.2^b	4.1 ± 0.3^ab	3.5 ± 0.2^a
Functional membrane Integrity (%)	52.1 ± 2.1^b	35.3 ± 4.1^a	30.4 ± 3.2^a
Viability after HOST (%)	72.1 ± 2.3	71.4 ± 3.5	62.2 ± 1.7

Pooled semen sample was diluted 1/3 in each of the sodium citrate – fructose solutions, pH 7.3 and incubated at 37°C in a water bath for 30 min prior to analysis. HOST: hypoosmotic swelling test; Number of replicates (n); 8, means bearing different superscript letters (a, b, c) within a row differ significantly (P < 0.05).

Effect of CEP-like osmolality in extender on quality attributes of ram sperm following liquid preservation

Tables 3–5 depict the effects of CEP-like osmolality in the extender on various sperm quality attributes following liquid preservation of ram semen. Irrespective of the period of storage, the straight-line velocity (VSL), linearity (LIN), straightness (STR) and beat cross frequency (BCF) were higher (P < 0.05), while the functional membrane integrity (FMI), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) were lower (P < 0.05) in semen preserved in hyperosmotic extender (390 mOsmol/kg; SL390) as compared to those preserved in the isoosmotic (360 mOsmol/kg; SL360) control. There were significant effects of interaction between the extender and storage period on sperm total motility (TM) and progressive motility (PROG); however, the interaction was non-significant on other parameters. At 48 h of storage, the TM, PROG, LIN, STR, and BCF were higher (P < 0.05) in the semen preserved in hyperosmotic extender compared to those preserved in the isoosmotic control. Similarly, at 72 h of storage, both the VSL and STR were higher (P < 0.05) while the FMI and ALH were lower (P < 0.05) in the semen preserved in hyperosmotic extender compared to those preserved in the isoosmotic control. The significantly higher values of the sperm quality attributes observed following 48 h but not 24 h of storage in the hyperosmotic extender compared to the isoosmotic control might be attributed to better protection of sperm qualities in the former extender while gradual deterioration of the same in the latter. However, both the sperm viability and acrosomal integrity were found similar (P > 0.05) between the two extenders over the period of storage. In contrast, at 48 h of storage both the PROG and VSL were significantly higher in the semen treated with hyper-osmotic extender compared to that treated with iso-osmotic extender, which suggested that CEP-like osmolality in the extender had beneficial effects on sperm motility and kinematics. Altogether, the above results suggested that the CEP-like osmolality in extender had little effect both on sperm viability and acrosomal integrity; however, it significantly improved the sperm motility and kinematics compared to the isoosmolality in the extender. The reduced HOS response (FMI) in sperm treated with CEP-like hyperosmotic extender compared to those treated with the isoosmotic control in the present study may not have represented the true functionality of plasma membrane, since both PROG and VSL were higher in presence of former extender and HOS reaction was reported to correlate positively with the sperm motility and viability (Jayendran et al. 1984). Rather, this decline in HOS response (FMI) might be attributed to the osmotic shock to the sperm resulting from the wider osmotic difference between the hypoosmotic solution and the hyperosmotic extender as compared to the isoosmotic control. The reduced HOS response obtained following pre-incubation of ram sperm in hyper storage, the TM, PROG, LIN, STR current study also supported the above notion. Altogether, the results suggested that ram spermatozoa were preserved better in hyperosmotic extender compared to isoosmotic extender. However, the exact mechanism by which higher osmolality in extender exerted beneficial effects on liquid preservation of spermatozoa is unknown. Probably, it caused osmotic extrusion of water from sperm thus increasing the ionic concentration in cytosol. The increased ionic concentration could have affected the sperm metabolism and hence the motility; which in turn might favor sperm preservation. The significant decline in mass motility following brief incubation of ram sperm in hyperosmotic solution (450 mOsmol/kg) observed in the previous experiment also supported the above notion. Although a few reports were available concerning the effects of osmolality of extender on liquid preservation of cauda epididymal sperm (De Pauw et al. 2003; Tamayo-Canul et al. 2011), the information on ejaculated semen is very scarce. To the best of our knowledge, the present study reports for the first time the effect of CEP-like osmolality of extender on liquid preservation of ejaculated ram semen. In contrast to the present study, a previous study reported that the motility of ram cauda epididymal sperm was significantly (P < 0.05) lower while the acrosomal integrity was higher following liquid-preservation in a hyperosmotic (420 mOsmol/kg) extender compared to a hypoosmotic (320 mOsmol/kg) extender (Tamayo-Canul et al. 2011). The difference between the two studies might be due to the differences in the experimental procedures. Because, in the current study, prior to motility assay the sperm samples were briefly incubated in an isoosmotic buffer (PBS containing 0.8% D-fructose, 330 mOsmol/kg) that might helped in revival of intracellular osmotic balance, sperm metabolism, and motility. However, in the previous study, no such sperm treatment was done prior to the motility assay; which probably had affected the revival of intracellular osmotic balance, sperm metabolism, and motility.

Table 3. Effect of cauda epididymal plasma-like osmolality of extender on sperm kinematics following liquid preservation of ram semen in soya lecithin-Tris-fructose extender at 3–5°C.

Sperm Attributes	Soya lecithin-Tris-fructose extender, pH 6.8 (mOsmol/kg)		Storage period (h)
	360	390	0	24	48	72
LIN (%)	39.5 ± 0.9^a	47.4 ± 0.4^b	45.4 ± 0.9	45.6 ± 0.5	42.9 ± 0.7	39.7 ± 1.9
STR (%)	74.0 ± 0.5^a	79.7 ± 0.5^b	77.4 ± 0.8	78.1 ± 0.6	76.1 ± 1.0	75.8 ± 0.9
VCL (µm/s)	175.1 ± 5.8^b	163.2 ± 6.3^a	211.0 ± 7.9^c	173.9 ± 5.4^b	149.4 ± 3.4^a	142.2 ± 4.3^a
VAP(µm/s)	94.9 ± 3.3	95.4 ± 3.6	119.8 ± 3.2^c	99.6 ± 2.8^b	82.7 ± 2.1^a	78.4 ± 2.4^a
VSL(µm/s)	71.3 ± 2.7^a	77.8 ± 3.1^b	94.8 ± 2.8^c	79.3 ± 2.4^b	64.2 ± 2.5^a	59.9 ± 2.1^a
ALH (µm)	8.0 ± 0.1^b	7.1 ± 0.1^a	8.3 ± 0.3^b	7.6 ± 0.1^a	7.2 ± 0.1^a	7.1 ± 0.1^a
BCF (Hz)	26.0 ± 0.5^a	28.6 ± 0.8^b	31.1 ± 0.5^c	27.4 ± 1.1^b	26.5 ± 0.6^ba	24.2 ± 0.8^a

Note: Interaction between extender osmolality and storage period was significant (P < 0.05) in TM and PROG. The interactions in the rest of the sperm attributes were non-significant. Values represent the mean ± S.E.M. of seven replicates. TM: total motility; PROG: progressive motility; LIN: linearity; STR: straightness; VCL: curvilinear velocity; VAP: average path velocity; VSL: straight-line velocity; ALH: amplitude of lateral head displacement; BCF: beat cross frequency. Means bearing different superscripts (a, b, c) within a row and factor differ significantly (P < 0.05).

Table 4. Effect of interaction between the extender and period of storage on sperm motility following liquid preservation of ram semen in soya lecithin-Tris-fructose extender at 3–5°C.

Treatment		TM (%)	PROG (%)
Soya lecithin-Tris-fructose extender, pH 6.8 (SLTF, mOsmol/kg)
	360	82.6 ± 1.3	55.9 ± 2.1
	390	82.6 ± 1.4	57.0 ± 1.9
	Overall	82.6 ± 1.3	56.4 ± 2.0
Storage period (h) SLTF Extender, pH 6.8 (mOsmol/kg)
0	360	89.2 ± 2.5	76.0 ± 2.3
	390	87.4 ± 2.7	75.1 ± 2.3
	Overall†	88.3 ± 1.8	75.8 ± 1.5
24	360	86.5 ± 2.1	69.0 ± 1.6
	390	87.3 ± 3.1	64.4 ± 2.7
	Overall	86.9 ± 1.8	67.6 ± 1.5
48	360	75.4 ± 1.5^a	39.5 ± 1.9^a
	390	83.3 ± 1.8^b	51.7 ± 2.3^b
	Overall	79.5 ± 1.3	45.6 ± 1.7
72	360	77.5 ± 2.1	37.4 ± 3.2
	390	70.3 ± 1.5	34.8 ± 1.9
	Overall	75.0 ± 1.5	36.1 ± 1.8

†Overall mean value at a given period of storage. TM: total motility; PROG: progressive motility. Means bearing different superscripts within a column and storage time differ significantly (P < 0.05).

Table 5. Effect of cauda epididymal plasma-like osmolality of extender on sperm membrane attributes following liquid preservation of ram semen in soya lecithin-Tris-fructose extender at 3–5°C.

Treatment	Viability (%)	Acrosomal integrity (%)	Functional membrane integrity (%)
Soya lecithin-Tris-fructose extender, pH 6.8 (mOsmol/kg)
360	94.5 ± 1.2	91.9 ± 0.9	75.4 ± 1.1^b
390	88.9 ± 1.7	90.1 ± 1.3	65.7 ± 1.5^a
Storage period (h)
0	95.3 ± 1.24^b	94.4 ± 0.83^b	75.7 ± 2.02
24	88.3 ± 1.25^a	90.7 ± 0.88^ab	70.6 ± 1.66
48	86.7 ± 2.00^a	90.2 ± 1.48^ab	70.6 ± 2.26
72	86.2 ± 2.29^a	88.3 ± 2.37^a	65.5 ± 2.00

Note: The interaction between the extender and storage period was non-significant (P > 0.05). Values are mean ± S.E.M. of seven replicates; means bearing different superscript letters (a, b, c) within a column and treatment differ significantly at P < 0.05.

Effect of CEP–like pH of extender on quality attributes of ram sperm following liquid preservation

The effects of CEP–like pH of extender on various sperm quality attributes following liquid preservation of ram sperm are presented in Tables 6 and 7. Irrespective of the period of storage, the sperm viability, acrosomal integrity, and FMI were similar (P > 0.05) between the SL6.8 and SL6.5 extenders. Similarly, both the sperm motility and kinematic attributes were also similar (P > 0.05) between the tested pH (pH 6.8 and 6.5) of the extender. There was no interaction between the extender and period of storage in any of the studied parameters. Acidic pH of cauda epididymal plasma and low intracellular pH (pH_i) has widely been considered responsible for sperm quiescence inside cauda epididymis. The pH_i of mammalian sperm is dependent upon the external pH (pH_e) and initiation of motility in cauda epididymal sperm is associated with rapid externalization of protons and increase in pH_i in presence of alkaline media (Hamamah and Gatti 1998). Hence, semen preservation may be improved by arresting sperm motility through use of acidic extender. However, the results in the present study indicated that sperm qualities were unaffected by use of cauda-like pH in extender during liquid preservation of ram sperm.

Table 6. Effect of cauda epididymal plasma-like pH of extender on sperm membrane attributes following liquid preservation of ram semen in soya lecithin-Tris-fructose extender at 3–5°C.

Treatment	Viability (%)	Acrosomal Integrity (%)	Functional membrane integrity (%)
Soya lecithin-Tris-fructose extender, 390 mOsmol (pH)
6.5	88.30 ± 1.61	62.70 ± 1.55	90.50 ± 1.19
6.8	88.90 ± 1.69	65.70 ± 1.48	90.10 ± 1.34
Storage period (h)
0	94.8 ± 1.46^b	94.1 ± 1.18^b	72.7 ± 1.73^b
24	90.0 ± 2.38^ab	91.0 ± 1.14^ab	66.5 ± 1.86^ab
48	84.7 ± 1.82^a	89.4 ± 1.44^ab	61.4 ± 2.43^ab
72	82.5 ± 1.92^a	85.7 ± 2.30^a	55.7 ± 1.36^a

Note: The interaction between the pH of extender and storage period was non-significant (P > 0.05). Values are mean ± S.E.M. of seven replicates; means bearing different superscript letters (a, b, c) within a column and treatment differ significantly at P < 0.05.

Table 7. Effect of cauda epididymal plasma-like pH of extender on sperm motility and kinematics following liquid preservation of ram semen in soya lecithin-Tris-fructose extender at 3–5°C.

Sperm Attributes	Soya lecithin-Tris-fructose Extender,390 mOsmol (pH)		Storage period (h)
	6.5	6.8	0	24	48	72
TM (%)	81.2 ± 1.4	79.4 ± 1.3	88.5 ± 1.5^c	80.9 ± 1.0^b	80.9 ± 1.5^b	68.9 ± 0.9^a
PROG (%)	57.2 ± 2.4	55.7 ± 2.5	76.5 ± 1.1^c	65.5 ± 0.7^b	59.8 ± 1.9^b	26.6 ± 1.6^a
LIN (%)	44.7 ± 0.5	45.0 ± 0.5	46.2 ± 0.4	44.8 ± 0.4	43.3 ± 0.8	45.0 ± 1.0
STR (%)	72.0 ± 0.6	73.0 ± 0.7	74.2 ± 0.7	71.4 ± 0.5	69.5 ± 1.0	74.3 ± 1.2
VCL (µm/s)	185.9 ± 10.0	187.0 ± 10.3	257.1 ± 4.8^c	188.9 ± 2.9^b	181.7 ± 8.1^b	118.2 ± 4.1^a
VAP(µm/s)	110.3 ± 5.9	110.7 ± 6.2	152.4 ± 3.0^c	112.6 ± 2.5^b	107.6 ± 4.0^b	69.3 ± 2.5^a
VSL(µm/s)	81.1 ± 4.6	81.5 ± 4.6	114.9 ± 2.1^c	81.9 ± 2.4^b	75.5 ± 2.4^b	52.7 ± 2.8^a
ALH (µm)	7.0 ± 0.2	7.3 ± 0.2	8.5 ± 0.1^c	6.8 ± 0.1^b	6.7 ± 0.2^b	6.3 ± 0.2^a
BCF (Hz)	15.3 ± 0.6	14.8 ± 0.8	17.3 ± 0.6^b	12.3 ± 0.8^a	12.7 ± 0.4^a	17.9 ± 1.0^b

Values represent the mean ± S.E.M. of seven replicates. TM: total motility; PROG: progressive motility; LIN: linearity; STR: straightness; VCL: curvilinear velocity; VAP: average path velocity; VSL: straight-line velocity. Means bearing different superscripts (a, b, c) within a row and factor differ significantly (P < 0.05).

In contrast to the present study, a previous study by Xu et al. (2009) reported that the motility of buck sperm was maintained better when the pH of the extender was adjusted from 6.6 to 6.04 during liquid preservation. The above difference between the two studies may be due to the narrower pH difference (pH 6.8 vs, 6.5) between the two extenders utilized in the current study as compared to the previous study.

In conclusion, the CEP-like osmolality in semen extender exerted beneficial effect on liquid preservation of ram sperm at 3–5°C. On the other hand, the extender having ram CEP-like pH (pH 6.5) was found comparable with the extender having pH 6.8 for liquid preservation of ram sperm.

Materials and methods

Materials and animals

All the chemicals used were of analytical grade and purchased from Sigma Aldrich, USA. Soya lecithin was purchased from Himedia, Mumbai, India. The osmolality of the extender was measured by an automatic cryoscopic osmometer (Osmomat 030, Gonotec, Berlin). The pH was measured by a digital pH meter (Eutech Instruments, Malaysia). Seven adult Patanwadi rams of 2–3 years of age were utilized in this study for collection of semen samples. The animals were maintained at the experimental animal farm of the division under semi-intensive system of management. An approval of the institute’s Animal Ethics Committee was obtained prior to the experimentation on animal.

Experimental design

In the present study, a total of four experiments were conducted to evaluate the effect of CEP-like osmolality and pH in semen extender on liquid preservation of ram semen. First, the physicochemical properties of ram CEP were analyzed to know the reference values in native sheep. Then, prior to conducting the experiment on liquid-preservation, an in vitro experiment was carried out to evaluate the effect of sperm treatment with hyperosmotic solutions on motility and functional membrane integrity. Finally, the effect of CEP-like osmolality and pH in extender on liquid preservation of ram semen was evaluated.

Experiment 1: Physicochemical properties of ram CEP

Ram CEP was isolated from cauda epididymis of 20 Malpura rams slaughtered at 9 months age in the livestock products technology section of the institute as detailed below. The pH and osmolality of CEP were measured by a digital pH meter (pH Tutor, Eutech Instruments, Singapore) and cryoscopic osmometer (Osmomat 030, Gonotec, Berlin), respectively. The concentration of Na⁺ and K⁺ were measured by a flame photometer (Systronics, Mumbai, India).

Experiment 2: Effect of brief incubation in hyperosmotic solution on mass motility and hypoosmotic swelling (HOS) response in ram sperm

To assess the effect of pretreatment of ram sperm with hyperosmotic solution on motility and functional membrane integrity freshly collected ejaculates from seven rams were pooled and diluted 1/3 in prewarmed sodium citrate-fructose solutions having osmolality 360, 390 and 450 mOsmol/kg (pH 7.3). Diluted semen samples were incubated at 37°C in a water bath for 30 min. Following incubation, both mass motility and hypoosmotic swelling (HOS) response were assessed following the procedure described by Evans and Maxwell (1987) and Jayendran et al. (1984), respectively.

Experiment 3: Effect of CEP-like osmolality in extender on liquid preservation of ram sperm

Semen collection, evaluation, pooling, and washing were done as detailed in general semen processing section. The basic extender was prepared by replacing egg-yolk with 2% soya lecithin in egg yolk-Tris-citrate-fructose (EYTF) extender (Mustafa 2009) and named as soya lecithin-Tris-fructose extender (SLTF). The iso-osmotic extender (360 mOsmol/kg, pH 6.8) consisted of 234.4 mM Tris (hydroxymethyl) aminomethane, 54.15 mM citric acid monohydrate, 44.4 mM D-fructose, 3 g/L strepto-penicillin (Dicristicin-S, Sarabhai Zydus, Mumbai, India) and 2% (v/v) soya lecithin prepared in double distilled water. The hyper-osmotic extender (390 mOsmol/kg) having cauda epididymal plasma-like osmolality (390 mOsmol/kg, pH 6.8) consisted of 168 mM Tris (hydroxymethyl) aminomethane, 51.6 mM citric acid monohydrate, 44.4 mM D-fructose, 59 mM NaCl, 30 mM KCl, 3 g/L strepto-penicillin (Dicristicin-S, Sarabhai Zydus, Mumbai, India) and 2% (v/v) soya lecithin. Following centrifugation as detailed below the sperm pellet was resuspended in corresponding extender and liquid-preserved at 3–5°C for 72 h. Semen samples were evaluated at 24 h intervals following treatment with 20% (v/v) SP at 37°C for 10 min.

Experiment 4: Effect of CEP-like pH of extender on liquid preservation of ram sperm

Semen collection, evaluation, and pooling were performed as detailed in the general semen processing section. The pooled semen sample was washed with extender having either CEP-like pH (pH 6.5) or normal pH (pH 6.8) by centrifugation as detailed in semen processing section. The pellet was resuspended in the same extender and liquid-preserved at 3–5°C for 72 h. The semen samples were evaluated every 24 h for various sperm quality attributes following pretreatment with 20% (v/v) SP at 37°C for 10 min.

The extender having CEP-like pH (pH 6.5 and osmolality 390 mOsmol/kg) consisted of 168 mM Tris (hydroxymethyl) aminomethane, 54.15 mM citric acid monohydrate, 40.0 mM D-fructose, 59 mM NaCl, 30 mM KCl, 3 g/L strepto-penicillin (Dicristicin-S, Sarabhai Zydus, Mumbai, India), and 2% (v/v) soya lecithin. The control extender (pH 6.8, osmolality 390 mOsmol/kg) consisted of 51.6 mM citric acid monohydrate, 44.4 mM D-fructose and rest of the components of SL6.5.

General semen processing and evaluation techniques

Isolation of ram CEP

Isolation of CEP was performed from cauda epididymis of 20 adult rams of 9 months age and Malpura breed that were slaughtered at the Livestock Products Technology Section of the institute. The CEP samples collected from the two epididymis of ram were pooled. Isolation of CEP was carried out following the procedure described previously (Paul et al. 2018a).

Semen collection and evaluation

Ejaculates were collected twice a week (Monday and Thursday) on seven occasions by using an artificial vagina (containing water at 39°C) and subjective evaluation was carried out as described by Evans and Maxwell (1987). Ejaculates having volume ≥0.75 ml, mass motility ≥4 (on a scale of 0–5) and concentration ≥3 × 10⁹ sperm/ml were pooled by taking equal volume from each sample to avoid individual variations.

Seminal plasma separation

Preparation of seminal plasma (SP) was performed from ejaculates collected from the same rams following the procedure described previously (Paul et al. 2018b).

Semen processing and storage

Pooled semen sample was diluted at 1/15 (v/v) with the respective extenders and washed by centrifugation at 150 × g for 10 min at room temperature. The supernatant was removed and the pellet was resuspended in the same extender at a final concentration of 800 × 10⁶ sperm per ml after determination through a hemocytometer. Aliquots of 2.5 ml volume were prepared in 5 ml glass vials having screw caps (Borosil, Mumbai, India). The vials were placed in holes made on a piece of sponge and the latter was properly fitted into a 250 ml glass beaker containing warm water at 37°C. The beaker was incubated in a cold handling cabinet, maintained at 5°C, for about 3 h until the temperature of water inside the beaker reached 5°C. Finally, the beaker containing the samples was shifted into a refrigerator, the temperature in which was present between 3ºC and 5ºC and stored for 72 h.

Sperm motility analysis by computer-assisted semen analyzer (CASA)

Prior to motility analysis semen sample was treated with SP as described before (Paul et al. 2018b). Sperm motility was analyzed by a computer-assisted semen analyzer (CASA, Hamilton-Thorn Biosciences HTM-IVOS version 12.1 M, Beverly, MA, U.S.A.) following the protocol and CASA settings described earlier (Kumar et al. 2007). The parameters measured were: curvilinear velocity (VCL, µm/sec), average path velocity (VAP, µm/sec), straight line velocity (VSL, µm/sec), % total motility (TM), % Progressive motility (PROG, VCL>25 µm/sec and straightness >80%), static (VAP<22 µm/sec), % linearity (LIN), % straightness (STR), % elongation (ELON, ratio of minor axis/major axis × 100), beat cross frequency (BCF, Hz) and amplitude of lateral head displacement (ALH, µm) of the spermatozoa.

Sperm viability

Viability of sperm was assessed by eosin-nigrosin staining as described by Björndahl et al. (2003). At least 200 sperm were counted randomly from five different fields at 400× magnification using a binocular microscope (Motic BE3 professional series, Tokyo, Japan).

Acrosomal integrity

Acrosomal integrity was studied by Giemsa staining procedure as described by Watson (1975) with brief modification as described previously (Paul et al. 2018b).

Functional plasma membrane integrity

The functional plasma membrane integrity (FMI) of ram sperm was assessed by performing hypoosmotic swelling (HOS) test as described by Jayendran et al. (1984) with little modification described previously (Paul et al. 2018b).

Statistical analysis

The mean ± standard error of the mean (S.E.M.) of the mass motility, sperm viability, acrosomal integrity, FMI, and motility parameters were determined. The values in percentage were subjected to arc sine square root transformation prior to the analysis. Data were analyzed by ANOVA using general linear model (GLM) multivariate repeated measures procedure of SPSS 16.0 (SPSS Inc. Headquarters, Chicago, IL, USA) and the effects of treatment, storage period and the interaction between the treatment and storage period were evaluated. The significance of the differences between the means was tested by performing Tukey post hoc analysis and considered statistically significant when P < 0.05. The mass motility score values were analyzed by Kruskal-Wallis ANOVA.

Authors’ contributions

Designed and conducted the experiment and drafted the manuscript: RKP; performed semen analysis: KB; analyzed the data: DK; revised the manuscript: RS.

Acknowledgments

The authors are thankful to the director of the institute for providing the necessary facilities to carry out this work. The authors also thank Mr Munir Ahmed for his technical assistance during the work.

Disclosure statement

No potential conflict of interest was reported by the authors.