Comparing Glycerol with Dimethylformamide and Combination for Cryopreservation of Pug Dog Semen in Egg Yolk Plasma Containing Extender

There is an increasing demand for preservation of canine semen among the breeders breeding specific breeds. Cryopreservation facilitates a wide use of ejaculates of stud dogs, irrespective of time and location. The success of the freezing and thawing of semen is dependent upon a number of steps expected to achieve less damage to the sperm and securing acceptable longevity in vitro and in vivo (Fastad, 1996). Many protocols have been described for canine semen cryopreservation and differences are linked to the composition of the extenders href="#peña_2003">(Peñaet_al2003) and nature of the cryoprotectants (Olar et al., 1989). Most of the researchers have used Tris-egg-yolk extender (Peña and Linde-Forsberg, 2000; href="#peña_2003">Peñaet_al2003). In order to find an alternative cryoprotectant for canine semen, different cryoprotectants, as dimethylsulphoxide and ethylene glycol, had been used either alone or in combination with glycerol (Cavalcanti et al., 2002; Martins-Bessa et al., 2006; Rota et al., 2006). The observations of those studies were based only on the analysis of post-thaw sperm motility or a few functional tests such as the hypoosmotic swelling test (HOST).  Some studies indicated that these cryoprotectants are similar (Martins-Bessa et al., 2006; Rota et al., 2006) or sometimes worse (Cavalcanti et al., 2002) than glycerol. Other alternative cryoprotectants for semen freezing are the amides, which have been frequently used as an option for equine semen cryopreservation (Alvarenga et al., 2005; Carmo et al., 2005). Amides have been also tested in canine semen freezing but with dissimilar observations on pooled semen samples of different breeds (Lopes et al., 2009; Oliveira et al., 2006; Mota Filho et al., 2011). 

Cryoprotective effects of amides are due to lower molecular weights (73.09) and viscosities in comparison with glycerol (92.05) and for higher membrane permeability. Since, addition of the methyl (CH3) radical into the amide molecule increases its permeability through the sperm membrane, therefore, it may improve the efficiency of its cryoprotective action (Bianchi et al., 2008). Therefore, the aim of the current study was to compare the effects of glycerol, DMF and their combination on motility, viability, plasma membrane integrity (PMI), acrosome integrity (AI), inner mitochondrial membrane potential (IMMP), reactive oxygen species (ROS) and antioxidant enzymes in frozen -thaw semen of pug breed.

Maintenance of dogs and semen collection
 
All the procedures were approved by the CPCSEA, New Delhi vide F. No 25-19-2018-CPCSEA, dated 22/11/2018. The experiments were conducted at Reproductive Biology Lab, Department of Veterinary Gynecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, Punjab, India from May, 2020 to October, 2020.
 
Analysis of sperm attributes in frozen-thaw semen
 
Semen was collected from four Pug dogs by digital method at an interval of 3 days. Motility was evaluated by wet mount and track method. Viability was assessed by eosin-nigrosin (Dhillon et al., 2020). Pooled ejaculates of four dogs were divided into three parts and extended with 1) Tris citric acid-fructose-egg yolk plasma-glycerol (TCFEYP-G), 2) TCFEYP-Dimethylformamide (TCFEYP-DMF) and 3) TCFEYP-G+DMF extenders and processed for cryopreservation as per standardized protocol in our lab (Cheema et al., 2021). AI of spermatozoa was assessed using Coomassie brilliant blue stain (CBB, R-250, Feng et al., 2007). Inner mitochondrial membrane potential was evaluated by staining the spermatozoa with JC-1 stain (JC-1 stain kit, Sigma -Aldrich, Cheema et al., 2021). About 200 spermatozoa were counted in different fields for all sperm attributes and percentage of motility, viability, plasma membrane integrity, acrosome integrity and IMMP [high (H), medium (M) and low (L)] was calculated.
 
Reactive oxygen species
 
Sperm extract was prepared in Tris buffer to evaluate following free radicles and antioxidant enzymes (Cheema et al., 2021).
 
Superoxide anion radical assay (Robak et al., 1988).
 
The reaction mixture was prepared by mixing 75 µl phosphate buffer (pH 7.4), 25 µl NBT (100 µM), 25 µl NADH (468 µM) and 25 µl sperm extract. The reaction was started by adding 25 µl phenazine methosulfate solution (60 μM) in to the reaction mixture followed by incubation at 25^oC for 5 min. A control was also run without sample. The absorbance was measured at 560 nm against blank. Percentage of SO₂ scavenging activity was calculated by using the following formula:
 

H₂O₂ radical assay (Ruch et al., 1989).
 
A solution of hydrogen peroxide (H₂O₂, 40 mmol/l) was prepared in phosphate buffer (pH 7.4). Reaction mixture was prepared by mixing 150 µl of 40 mmol H₂O₂ and 25 µl sperm extract and incubated at room temperature for 10 min. Absorbance of 40 mM H₂O₂ (control) and reaction mixture was measured at 230 nm against PBS, pH 7.4. H₂O₂ activity (%) was calculated by the following formula:
 

 
Nitrite radical assay (Marcocci et al., 1994).
 
Griess reagent (50 µl) and sperm extract (25 µl) were mixed and incubated at room temperature for 10 mins. A blank consisting of 50 µl PBS, pH 7.4 and 25 µl sperm extract was also ran along with the samples. The absorbance of the chromophore that formed during diazotization of the nitrite with sulfanilamide and subsequent coupling with 1-naphthyl ethylene diamine dihydrochloride was immediately read at 540 nm. NO₂- concentration in the samples was calculated from standard curve plotted for 1mM-10 mM concentrations of sodium nitrite.
 
Lipid peroxidation and antioxidant enzymes
 
Malondialdehyde concentration (end product of LPO), Glutathione reductase (GRE), Glutathione peroxidase (GPx) and Catalase (CAT) were estimated in sperm extracts as per modified and standardized methods in our earlier study (Cheema et al., 2021). MDA concentration and enzyme activity were calculated by using the following formula:
 

 

 
GRE (IU/10⁹ spermatozoa/min) =   ΔT - ΔC
GPX (IU/10⁹ spermatozoa/min) =   ΔT - ΔC
Where,
ΔT - Change in OD _Test at 60 sec interval and ΔC - Change in OD _Control at 60 sec interval.
 

Comparative evaluation of extenders based on sperm attributes
 
Values for motility, viability, PMI, AI were significantly (p<0.05) high in TCFEYP-G compared to TCFEYP-DMF and TCFEYP-G+DMF at post thaw. However, HIMMP and MIMMP/MDA were non-significantly (P>0.05) high and low in TCFEYP-G compared to TCFEYP-DMF and TCFEYP- G + DMF at post thaw, respectively (Fig 1). There was a difference of 23.4%, 21.2%, 19.4%, 14.7%, 15.0%, 8.7%, 40.1 µM and 21.7%, 24.1%, 7.7%, 24.7%, 12.5%, 21.2%, 42.5 µM in motility, viability, PMI, AI, HIMMP, MIMMP and MDA concentration between TCFEYP-G x TCFEYP-DMF and TCFEYP-G x TCFEYP-G + DMF extenders, respectively. Higher motility, viability, PMI, AI, HIMMP and low MIIMP/Lipid per oxidation (MDA production) in TCFEYP-G extender indicated that glycerol gives more protection to the sperm against cryo-effect than DMF and combination of both. A study on canine semen indicated significant difference (P<0.05) between glycerol and DMF with regard to subjective progressive motility, objective progressive motility, velocity average pathway and amplitude of lateral head, which also confirmed the efficiency of glycerol (Lopes et al., 2009) for cryopreservation of canine semen. Overall, sperm motility and membrane intactness/function were higher when glycerol was used as a cryoprotectant, as compared to DMF (P<0.05) (Hernandez-Aviles et al., 2020).



Penetrating and non-penetrating Cryoprotectants (CPAs) reduce the physical and chemical stress exerted by the freezing process on spermatozoa. The most frequently used cryoprotectant for dog semen cryopreservation is glycerol which has a mixed intracellular and extracellular action. Glycerol reduces the salt concentration, thus lowering the freezing point of the solution and the risk of membrane perforation. It can interact by hydrogen bonding with water and can permeate across the plasma membrane of many different cell types at a relatively slow rate. Membrane permeability of DMF is higher in comparison to glycerol (molecular weight 92.05) due to its lower molecular weight (73.09) and viscosity. In general, permeability decreases as the molecular size of the substance increases. CPAs with low permeability can cause more osmotic stress than CPAs with high permeability. Although, glycerol has a high molecular weight compared to DMF, but there was low level of cryoinjury in TFCEYP-G compared to TFCEYP-DMF and combination of both extenders. It may be revealed from this study that there was no significant advantage using DMF to replace glycerol in cryopreservation of canine semen. Our observations are in agreement with that reported in canine (Futino et al., 2008; Lopes et al., 2009; Hernandez-Aviles et al., 2020). However, it is suggested that 5% DMF in lactose extender could efficiently preserve post thaw quality of canine semen, with motile sperm values of 45.0% (Oliveira et al., 2006). DMF at a concentration of 7% was added to the Tris base extender in the present study. In earlier studies, concentration of DMF and buffer were different (Oliveira et al., 2006; Futino et al., 2008; Lopes et al., 2009). It may be assumed that when DMF is used to cryopreserve canine semen, the concentration (Pena et al., 1998), method (Silva et al., 2003) and temperature (Silva et al., 2006) of its addition may affect the quality of frozen-thawed sperm.
 
Comparative evaluation of extenders based on lipid per oxidation, free radical scavenging and antioxidant enzyme activity
 
MDA concentration (µM/109 spermatozoa) was significantly (p<0.05) low in TCFEYP-G (47.7±1.3) compared to TCFEYP-DMF (96.6±17.5) and TCFEYP-G + TCFEYP-DMF (102.6±3.2) extenders (Fig 2). However, H2O2, scavenging capacity of spermatozoa, cryopreserved in TCFEYP-G (90.5±0.4%) was significantly (p<0.05) higher than in TCFEYP-DMF (70.9±6.0%) and TCFEYP-G + TCFEYP-DMF (81.3±1.7%) (Fig 2). However, there was no significant (p>0.05) difference in superoxide free radical scavenging activity and nitrite concentration of spermatozoa among the three extenders (Fig 3). Activity of GPX, SOD and Catalase was also significantly (p<0.05) higher in TCFEYP-G (0.34±0.06, 144.2±1.0, 471±5.5 IU/109 spermatozoa) compared to TCFEYP-DMF (0.27±0.08, 149±2.3, 451.3±3.2 IU/109 spermatozoa) and TCFEYP-G+DMF (0.07±0.02, 137.3±1.7, 384±4.5 IU/109 spermatozoa) (Fig 2,3). However, values were reverse for GRE activity (Fig 3).

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Studies conducted in other animals, also have demonstrated a reduction in the sperm membrane intactness following semen cryopreservation using DMF as a cryoprotectant (Bezerra et al., 2011 and Forero-Gonzalez et al., 2012). It may be assumed from poor results yielded using DMF as a cryoprotectant that the sperm plasma membrane from those species has a similar composition to the canine sperm membranes and hypothesized that a common susceptibility to low-molecular weight cryoprotectants could exist in these species (Hernandez-Aviles et al., 2020). The presence or absence of different transmembrane proteins with affinity for a specific cryoprotectant might account for the species-specific response to the effect of DMF or glycerol during freezing. Horse sperm cryopreserved in extender containing DMF and with better tolerance to DMF exhibits no difference in the relative expression of Aquaporin-7 (AQP-7). On the other hand, the use of this same extender was related to a differential expression of AQP-3 and AQP-11 (Bonilla-Correal et al., 2017).

In other species, where semen freezing relatively goes well with amide-containing extenders (boars and bulls), the differential expression of these water and glycerol transporting proteins has also been demonstrated (Prieto-Martínez  et al., 2017a; Prieto-Martínez  et al., 2017b). It can be assumed from these observations that the transport of amides could be differentially affected depending on the type of cryoprotectant used in the extender, which in turn may be differentially related to the presence of aquaporins in canine semen. Lower osmotic coefficient of DMF may be another reason that allowed its faster passive diffusion through the plasma membrane, which reduced the amount of cryoprotectant available within the sperm before freezing (Oldenhof et al., 2012).

Deterioration of PMI, AI and HIMMP in DMF containing extender compared to glycerol correspond to the motility and viability during the present study. It indicated that HOST and HMMP were the parameters which explained the variability in post-thaw motility observed after freezing (Hernandez-Aviles et al., 2018). The HOST predicts the function of the sperm membrane by evaluating the capacity of the sperm cell to react to an osmotic challenge when is incubated in a low-osmolality solution (Ramu and Jeyendran, 2013) and a higher mitochondrial membrane potential is associated with a more efficient energy production through the mitochondrial electron transport chain (Amaral et al., 2013). Further deviant mitochondrial function is associated to oxidative stress and apoptosis (Aitken et al., 2016). MDA production indicating oxidative stress was high, while H₂O₂ scavenging activity and SOD, catalase, GPX enzyme activity of spermatozoa were low in DMF containing extender compared to glycerol. It may be predicted that sperm plasma membrane osmotic capacity is reduced when the plasma membrane intactness or the mitochondrial electron transport chain are compromised due to osmotic stress or oxidative stress (Gonzalez-Fernandez et al., 2012 and Ortega-Ferruso et al., 2017). This compromise was more in spermatozoa cryopreserved in DMF containing extender. Higher percentage of free radical scavenging activity and higher activity of SOD, GPX and catalase in semen, cryopreserved in TCFEYP-G revealed that balance of antioxidants and free radical formation was better maintained by glycerol than DMF or their combination. Therefore, higher percentage of motile, viable spermatozoa, higher intact plasma membrane, intact acrosome and high potential of mitochondrial membrane were a result of reduced oxidative stress in TCFEYP-G extender. Excessive production of free radical is responsible for alterations on semen quality, caused by decrease in sperm motility, as well as protein and DNA damage, resulting in cell apoptosis (Lucio et al., 2016).

To our knowledge, this is the first study in which the effect of DMF on the membranes of dog spermatozoa is evaluated on a combination of sperm function test including oxidative stress, ROS and antioxidant enzymes.

This study concludes that glycerol is a healthier cryoprotectant than dimethyl formamide to protect canine spermatozoa from cryo-injury during freezing-thawing process of canine semen.

All authors declare that they have no conflict of interest.