#veterinary sonography

Abstract

Evaluation of the concentrations of the sperm macromolecule called proAKAP4, has been successfully introduced as a pertinent sperm parameter to assess sperm quality and high concentrations of proAKAP4 was shown to be highly correlated with sperm motility and fertility in large mammals. The sandwich ELISA kits known as Pig 4MID® Kits allowed the artificial insemination stations to monitor sperm quality more accurately with threshold values qualifying each ejaculate and animal. Introducing new methods and procedures are always challenging and sperm frozen collections have been suggested to standardize sperm assessment in daily routine. We thus have designed an experimental study to assess proAKAP4 stability and integrity in neat frozen ejaculates. Following baseline measurements, fresh ejaculates were aliquoted and stored at -20 °C for stability experiments up to 6 months and following up to 10 freeze-thawing cycles. ProAKAP4 concentrations were assayed at each time point using the Pig 4MID® Kit and western blot. Median or mean changes from baseline concentrations were evaluated statistically. We showed that the frozen storage conditions neither modified the total proAKAP4 concentrations nor changed the degradation rates of the proAKAP4 into mature AKAP4, that should in turn ensures signaling, capacitation and motility. This sperm parameter was shown then to be robust for semen quality analysis on fresh and on frozen neat semen. Taken together, proAKAP4 polypeptide can be considered as a highly stable analyte when kept frozen in raw semen up to the semen quality analysis using the Pig 4MID® Kit

Keywords: Boar; Proakap4; 4MID®; Fertility; Stability; Precursor; Freeze-thaw cycle; Storage; Semen processing

Abbrevations: AKAP4: A-kinase Anchor Protein 4; PKA: Protein Kinase A; CASA: Computer Assisted Semen Analysis

Introduction

ProAKAP4 concentrations are considered as a new sperm parameter that have been validated by field studies for sperm analysis assessment in large mammals [1-6]. Measurement of proAKAP4 concentrations was thus reported to generate pertinent information to guide the prognosis of sow fertility and prolificity in highly competitive breeding activities [1,4]. This quantitative approach of semen assessment is based on a sandwich ELISA method that allow to compare up to 87 semen simultaneously and is commercialized under the brand name of the 4MID® Kits (4BioDx, France). The Pig 4MID® Kits provide then a reliable and valuable figure reflecting the amount of proAKAP4 in pig ejaculates, with threshold values that are allowing a follow-up of the sperm quality inside and between pig breeding centers. Structurally, proAKAP4 is a precursor protein and will have to be converted by motile and alive spermatozoa in AKAP4 (A-kinase anchor protein 4) that in turn, coordinate the main transduction signals regulating sperm motility, capacitation and fertility [1,7-11]. ProAKAP4 concentrations has been reported to be correlated with total and progressive motility in stallion, in human and in bull [2,3,6,12,13]. Clearly the proAKAP4 concentrations is a reflect of the sperm motility giving a more objective figure compared to microscopic observations of the spermatozoa that are motile only at the time of analysis. In contrast, with the Pig 4MID® Kit, the more the proAKAP4 concentration is high in the ejaculate, the more the spermatozoa will be motile and efficient to go up to the site of fecundation. They have been evidences that spermatozoa with few or without proAKAP4 will be less motile or immotile and then infertile [14-18]. Therefore, we considered as essential to determine the stability and integrity of the full-length proAKAP4 in frozen storage conditions before the critical step of the sperm quality analysis. Data concerning the effects of freezing, thawing, and long-term storage effect on sperm proAKAP4 concentrations were not yet available in the literature. In this study, we aimed then to examine the analytical stability of proAKAP4 in fresh boar semen. We then assess the variations of proAKAP4 concentrations and proAKAP4 degradation rates in following freeze-thaw cycles and in long-term storage at minus 20 °C, in a final goal to improve operating procedures for semen analysis in swine breeding centers.

Materials and Methods

Sperm Preparation

Fresh boar sperm samples (Large White strain) were obtained from a boar stud and was first checked for total volume. They were then aliquoted into 1.5-mL polypropylene cryovials for the stability experiment. For stability assessment, the sampling of each ejaculate was then composed of 5 aliquots (2 for freezethaw cycle experiment and 3 for long-term storage experiment). Following baseline measurement (T0), they were all maintained frozen until analysis. The remaining boar ejaculates were either processed for the control quality experiment or for proAKAP4 expression controls. Samples stored at -20 °C were kept in a freezer equipped with a temperature recorder.

Freeze thaw Cycles and Long-term Storage Experiments

After 24 hours, 2 frozen sperm aliquots were thawed at room temperature until completely thawed, and then mixed properly with a micropipette before analysis (freeze-thaw 1). Samples were immediately re-frozen at -20 °C. This cycle was repeated for ten consecutive time points (T1, T2, T3, T4, T5, T6, T7, T8, T9, T10) to yield freeze- thaw processing. A group of 3 semen aliquots were stored at -20 °C for up to 1, 3 and 6 months, and then analyzed for stability at three-time intervals (T1M, T3M, T6M). As described below, the concentrations of proAKAP4 were assessed at each time point using the Pig 4MID® Kit (4BioDx, France). In parallel, proAKAP4 expressions and metabolism of the same aliquot were examined by western blotting. The results were compared with those obtained from the initial analysis of fresh samples. Median or mean changes from baseline (T0) concentrations were evaluated statistically.

Spermatozoa and Seminal Plasma Preparation from Boar Semen

A volume of 500μL of the remaining fresh semen was added in a 1.5mL Eppendorf tube and then centrifuged during 10 min at 2000rpm. The supernatant over the spermatozoa pellet was recovered with a 200μL micropipette and corresponded to the seminal plasma fraction. One volume of Tris Buffer (10 mM Tris HCl pH 6.8) with 2% SDS was added to the seminal plasma and sonicated at 22kHz (15 Watts) for 30 seconds. In parallel, 250μL of Tris Buffer with 2% SDS was added to the spermatozoa pellet, mix thoroughly with a vortex and then sonicated for 30 seconds (22 kHz, 15 Watts). Protein concentrations were determined using the Bradford’s method (BioRad, France). Then 50μL of the Tris-SDS sample was added to 1 volume of 2x concentrated NuPAGE LDS Sample Buffer (ThermoFisher, USA) and 10μL of NuPAGE Sample Reducing Agent (ThermoFisher, USA). Samples were vortexed and heated at 80 °C for 10 min.

Analysis of pig ProAKAP4 Expression and Metabolism by Western Blot

Equal protein concentrations of each sperm preparation were loaded on polyacrylamide gel (4-12% NuPage Precast Gels) and then transferred onto 0.45μm nitrocellulose membranes (G&E Healthcare, USA) using the Liquid Transfer System (Life Technologies, USA). Membranes were incubated overnight at 4 °C with the first antibody at a dilution of 1:4000 in 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% (v/v) Tween 20 (TNT Buffer), either with the clone 7E10, a monoclonal antibody anti-AKAP4 (4BioDx, 4BDX-1602, France), or with the clone 6F12, a monoclonal antibody anti-proAKAP4 (4BioDx, 4BDX-1701, France). After washing 3 times in TNT (10 min), each membrane was incubated with the secondary anti-mouse antibody coupled to horseradish peroxidase at 1:50000 diluted (Vector Laboratories, Burlingame, CA USA) and revealed with the ECL™ chemioluminescence kit (G&E Healthcare, USA). Images were acquired using the Image Quant™ LAS 4000 system (G&E Healthcare, USA).

The Pig 4MID® ProAKAP4 ELISA Assays

Thawed semen samples (respectively 50, 25 and 12μL) were mix with the Pig Lysis Buffer (450, 475 and 488μL) and then proceeded for ELISA quantification using the Pig 4MID® Kit (4VDX-18K2) according to the manufacturer’s instructions (4BioDx, France). Briefly, 100μL of semen lysates was then added to each well of the antibody-coated plate. A solution with conjugated proAKAP4 antibody was then added and after appropriate washing, the complexed sandwich was incubated with a substrate solution. The resulting color intensity was proportional to the amount of proAKAP4 present in each semen sample and could be measured by spectrophotometry at 450nm. A standard curve was determined in parallel for precise concentrations of proAKAP4 in the pig semen sample. Results of proAKAP4 concentrations were always expressed in ng/mL.

Statistical Analysis

Statistical analysis was achieved using Prism 8.2 GraphPad software (GraphPad Software, USA). D’Agostino and Pearson normality tests were performed to determine if the populations were following a Gaussian distribution and Pearson correlation coefficients were determined for each proAKAP4 concentration. The threshold for statistical significance was set to be p<0.05. In normally distributed groups, results were presented as mean ± standard deviation. The significant differences from T0 value were determined by a non-parametric paired samples t-test Mann Whitney U-test. Stabilities of proAKAP4 after freeze thaw cycles and after long term storage were assessed by the percentage change from T0 for paired groups (T0-T1, T0 -T2, etc. and T0 – T1M, T0 -T3M, etc.). Bias was calculated by the formula: [(CX - C1)/C1] × 100%, with C1: the mean or median of the T0 sample; and Cx: the mean or median of the experimented sample. For non- Gaussian groups, median variations from T0 were determined by non-parametric Friedman test and Wilcoxon signed rank test

Results

ProAKAP4 Expression in Boar Raw Semen

As observed previously from other mammals [1-3] proAKAP4 was only expressed in spermatozoa preparation and not in the seminal liquid as revealed with the monoclonal antibody (clone 6F12) against proAKAP4 (Figure 1). The proAKAP4 was cleaved in AKAP4 mature protein and the prodomain was released (Figure 1A). This cleavage and metabolism of the precursor proAKAP4 can also be followed by western blotting using specific monoclonal antibodies such as the clone 7E10 which recognized the C-terminus of both proAKAP4 and AKAP4 (Figure 1B). Therefore, in this initial T0 experiment, we observed the same amount of proAKAP4 and AKAP4 in the spermatozoa preparation sample of the fresh pig ejaculate. As expected, we confirmed that proAKAP4 is a spermatozoa specific protein expressed in the flagellum of pig spermatozoa

Stability of Boar proAKAP4 during Freeze-Thaw Cycles of the Same Aliquot

The concentration of proAKAP4 was measured in the ejaculate using the Pig 4MID® Kit as T0 value for the stability experiments. The initial mean concentration of ProAKAP4 was of 50.7 ± 1.3ng / mL, reflecting a high-quality semen [1]. After semen aliquots have been frozen and thawed up to ten times, there were no statistically significant differences in proAKAP4 concentrations as quantified using the Pig 4MID® Kit from T0 to T10 (Table 1).

All concentrations were in ng /mL and indicated as a mean± SD and median (interquatile ranges). Clearly, the proAKAP4 concentrations were not modified statistically after ten freezethaw cycles and the global percentage of variations was at 9.54%. Dilutions of the neat semen (half and quarter dilution factor) had no effect on the recovery of proAKAP4 concentrations as shown graphically on Figure 2. These dilutions highlighted the robustness of the Pig 4MID® Kit to quantify accurately the amount of the proAKAP4 polypeptide in neat pig semen. We checked then the expression and metabolism of proAKAP4 by western blotting (Figure 3). None of proAKAP4 and AKAP4 expressions or metabolisms were altered by the freeze-thaw cycles. Neither the integrity of proAKAP4 or AKAP4 was shown to be altered along the 10 freeze-thawing cycles and proAKAP4 was not further converted into AKAP4 showing that proAKAP4 and AKAP4 processing were not modified by freeze thawing cycles. The proAKAP4 was therefore considered as a very stable analyte when kept frozen in raw semen until we performed the Pig 4MID® Kit analysis

Stability of the Frozen proAKAP4 Polypeptide in longterm Storage Conditions

They were no significant variation in proAKAP4 concentrations as measured with the Pig 4MID® Kit for fresh pig sperm when stored until 6 months at -20 °C (Table 2). No variations were obtained when stored at - 80 °C (data not shown). Statistical significances were evaluated as described in the Materials and methods section. Our results showed that total proAKAP4 concentrations were clearly stable up to six months of storage at -20 °C with the variation in proAKAP4 concentration always below 5%. The western-blot analysis displayed no degradation of the sample stored at -20 °C from up to 6 months highlighting the robustness of the protein when kept frozen in raw semen (data not shown).

Intra-assay and Inter-Assay of the Pig 4MID® Kit

We further assess the robustness of the Pig 4MID® Kit by evaluation of the intra-assay and inter-assay CV’s on the Pig 4MID® Kit with neat pig semen as in the design of our study. These intra-assay and inter-assay CV’s were performed with two different ejaculates of the same animal (Table 3). Inter-assay variation was assessed from 10 determinations (with 2 aliquots each day) on ten consecutive study days, and intra-assay variation was calculated from eight sequential determinations obtained from the first day of the study period

Discussion

This study examined the storage effects and repeated freezethaw cycles on pig proAKAP4 sperm protein integrity in preanalytical conditions (meaning before the 4MID® Kit procedures) to evaluate the robustness of this new parameter in daily routine of semen analysis for swine breeding activities. We clearly show that proAKAP4 polypeptide is highly stable when frozen at minus 20 °C, for a long time period (up to 6 months) and will not be altered by multiple freeze-thaw cycles in neat semen. These data are of importance as they highlighted for the first time, that specimens of one ejaculate can be aliquoted and kept at minus 20 °C until their analysis and shipped from AI stations to central laboratories without loss of proAKAP4 integrity.

The reason of this stability could be due to the localization and the inherent functionality of the proAKAP4 itself. As shown on Figure 1, the proAKAP4 is a sperm specific protein that is neither found on the membrane nor released in the seminal plasma. The proAKAP4 polypeptide is inside the spermatozoa, more precisely in the fibrous sheath of the principle piece of the flagellum [19-22] and will need to be released from the fibrous sheath to be further quantified using the 4MID® assay. ProAKAP4 has been shown to be strictly localized to the principal piece of the flagellum and not in other spermatozoa compartments [20-21], tightly anchored to the fibrous sheath, along the longitudinal columns and ribs of the sperm tail [2,3,20-21].

According to the Pig 4MID® assay procedure, the proAKAP4 has then first to be extracted from the spermatozoa. Proteins markers described in sera or in seminal fluids [1,23] are frequently reported to suffer from the shear stress induced in buffered solutions and from long-term storage conditions. In contrast of what we reported with sperm proAKAP4, proteins in buffer solution can be fragile and they may even acquire conformations susceptible to degradation during frozen and post-thawed conditions. Clearly, proAKAP4 concentrations appears to be stable as long as the polypeptide is maintained in neat semen within the spermatozoa flagellum, with the fibrous sheath bringing stability for proAKAP4 integrity. The maintenance of proAKAP4 as a fulllength precursor is then important for the aliquot processed for the initial quality assessment of the ejaculate and at further steps, for the quality control during dose processing in AI stations. High proAKAP4 concentrations in the ejaculate and then in doses, will ensure to have enough motile and functional spermatozoa populations in the hours post the artificial insemination

The total amount of proAKAP4 per spermatozoa is fully synthetized within the testis and before ejaculation. Therefore, an aliquot of the ejaculate could be frozen immediately after semen collection in boar studs as this will represent the exact picture of the long-term motility of the spermatozoa. Freezing of an aliquot of ejaculate at collection point will then facilitate the analysis of semen (related to the proAKAP4 concentration) and favors also transport of such aliquot up to external laboratories. Our results clearly showed that degradation rates of the proAKAP4 were not impacted by frozen storage conditions of the aliquot and are in favor of such collection for delocalized sperm quality assessments. Furthermore, proAKAP4 stability when stored in aliquots in sperm frozen collections, will allow to better take in account technical and logistical constraints such as i) delays in shipping frozen aliquot when in need to analyze hypofertile animal; ii) being less dependent of any power cut or voltage fluctuations of the low-cost freezers; or the use of frost-free freezer that goes through numerous defrost cycles, as may happen in small breeding centers.

In boar stud, the storage of frozen aliquoted samples could also be convenient to process all the semen in the same time to compare ejaculates of different animals at the end of collection time. The dose semen processing will then not be impacted as the 4MID® analysis will be completely run in 2 hours. The amount of proAKAP4 as a read out of sperm quality should add marketing values for AI stations by ensuring high quality semen. In swine industry, there is also a real interest to identify the best male and then to follow up the sperm production during exploitation. Boars are usually kept from 6 to 9 months in the AI stations. That will be of importance to have a stable parameter to follow animal along all his career and keeping a safe measure of the initial quality of the first ejaculate after quarantine. In this context, the storage of frozen aliquoted samples may allow likewise to identify genetical traits of interest in a particular pig strains, such as fertility, death at birth or litter size, that may be related to proAKAP4 levels of expression [1,24-27].

Finally, keeping frozen aliquots of pig semen could allow to reanalyze the same samples stored to confirm previous results or to perform additional analysis, establishing new path for boar sperm preservation investigations. Better understanding proAKAP4 stability allows now to compare ejaculates at different collection points and compared to extended semen which is being shipped and used many days later. The storage capacity of extenders should be then further explored in relation with proAKAP4 consumption and degradation rates, during several days and in chilled conditions, when spermatozoa will stay alive.

Conclusion

One of current challenge of the swine industry is to standardize the semen processing procedures within boar studs. The proAKAP4 parameter have been initially introduced to facilitate the identification of ejaculates of inferior motility and quality, that were not identified by classical sperm parameters, and that could then be withheld before their release into the field. Having a stable sperm parameter such as proAKAP4 that can be kept stable as frozen up to the analysis time should be further interesting for quality check control and to follow up this parameter evolution during all the boar career. Taken together, the proAKAP4 parameter stability present then multiple advantages in favor of harmonizing sperm quality assessment between laboratory and AI centers.

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Abstract

Animal body reacts to all kinds of injuries and stress to keep up the homeostasis mechanism of the body. This homeostasis achieved either by or nonspecific mechanism. The nonspecific innate resistance of the body like cytological and cytokine reactions including fever, leukocytosis etc. is known as acute phase response. In this response, there will be increase or decrease of serum concentration of proteins. These proteins are known as acute phase proteins. Measurements of serum concentration of these acute phase proteins are found to be useful in assessment of health status and prediction of diseases of the man and animals. The serum concentration of these acute phase proteins returns to base levels when the triggering factor is no longer present. The acute phase response is now considered to be a dynamic process involving systemic and metabolic changes providing an early nonspecific defence mechanism against insult before specific immunity is achieved. Use of one of the acute phase proteins, C-reactive protein as biomarkers for animal disease diagnosis and health status assessment has got high potential in modern veterinary practice is discussed in this review

Keywords: Acute injury; C - reactive protein; Acute phase proteins; Veterinary practice

Abbrevations: CRP: C Reactive Protein; Hp: Haptoglobin; AGP: Acid Glycoprotein; SAA: Serum Amyloid A

Introduction

The acute phase reaction encompasses all the phenomena which take place in animals following tissue damage and is particularly associated with inflammation from whatever cause. During the acute phase reaction, the body mounts a multifactorial response to remove and replace damaged tissue and one of the mechanisms involved is the production and secretion by the liver of a number of ‘acute phase proteins’ [1]. The concentrations of these proteins increase during the reaction are called as Positive acute phase proteins (APPs) such as C reactive protein (CRP), Serum Amyloid A (SAA), Haptoglobin (Hp), Ceruloplasmin, α2- Macroglobulin, α1 Acid Glycoprotein(AGP), Fibrinogen and Complement (C3,C4) while those of others, including albumin, Transferrin, Transthyretin and Retinol-binding protein decrease as the liver switches production of protein towards the synthesis of the proteins required to deal with the damage; is called as negative APPs [2] (Figure 1).

Biological functions of C-reactive Protein

The protein was named the C-reactive protein because of its ability to bind pneumococcal C-polysaccharide. The presence of CRP has also been described in human patients during acute infections caused by acute lobar pneumonia, active rheumatic fever and bacteraemia caused by “colon bacillus”. Among the biological functions described in the literature are Complement activation and opsonisation [3,4] Modulation of monocytes and macrophages, cytokine production [5] Binding of chromatin [6] Prevention of tissue migration of neutrophils

CRP in Bovines

During the early stages of infection, the serum concentration of CRP increases [3]. This increase has been described to be evident before an elevated rectal temperature is observed. Even though increased concentrations of bovine CRP during naturally occurring infections and a correlation with herd health status have been reported, CRP is generally not considered an acute phase protein in cattle. As stress increases to a critical point, the liver rapidly synthesizes large amounts of CRP and releases it into the blood to provide immediate protection against stress [7]. Diseases in a dairy herd elevated the serum CRP level. The serum CRP level was also correlated with milk production. The greater the milk production, the higher the level of serum CRP. Diseases, especially acute infections, induced much higher levels of CRP production than stress or lactation. Also showing that plasma C-reactive protein concentration is related to different kind of stress (poor health, high lactation, blood collection). Strong correlation was observed in cows after delivery (0-1 month) between fibrinogen and CRP values [8]. Obtained results suggest that not only inflammations but also physiological factors such as pregnancy, delivery and/or state of lactation may have a significant impact on APPs values in the blood plasma of dairy cows. It would be worth in the future to check whether there is a relationship assessing the animal health status obtained using acute phase proteins method relatively to other indicators, such as milk yield, length of lactation or others. Morimatsu [9] also discovered Elevation of bovine serum C-reactive protein by lactation when compared to other Acute Phase protein such as serum amyloid P component levels (Figure 2).

CRP in Swine

In the pigs, as in the dogs and humans, C-reactive protein (CRP) is the prototypical acute phase protein with major diagnostic value. CRP concentrations are useful for evaluating the health status of a swine herd, but not for the health status of an individual animal or the differentiation of diseases. Serum haptoglobin (HP) concentration is better than serum CRP concentration as an indicator of inflammatory reactions in pigs, and HP is an important marker for swine health status [10]. On the other side, pigs undergone experimental study had CRP serum concentration below 22μg/ml (mean 18.64 ± 2.59). Twenty-four hour after coinfection with swine influenza virus (H1N1) and Pasteurella multocida, the mean concentration of CRP reached 62.85 ± 35.55μg/ml. Significant difference were noticed as compared to control animals [11]. The presence of elevated serum Hp and CRP concentrations in apparently healthy pigs at slaughter could provide important information to a veterinary inspector about the presence of sub-clinical lesions that could lead to condemnations or a decrease in the quality of carcasses [12].

CRP in Canines

In canines CRP is the major APP used as marker for systemic inflammation / infection. Normally the level of CRP is less than 1.5 mg/ dL or even lower than 0.5 mg/dl. The normal range may be 0.08 to 2.26 mg/dl [13]. The level rises within 4 to 6 hrs after onset of inflammation / infection. Serum CRP level above 3.5 mg/dl, indicates presence of systemic inflammation. Level above 5 mg/dl is a strong evidence of systemic inflammation. Strong correlation was observed between CRP and animal’s temperature and Total Leukocyte counts of canine patients naturally infected with Leptosporosis [14]. CRP levels could be used to monitor early responses to antibiotic treatment and might alert veterinarians to the need for further evaluation or additional treatment. Serum CRP concentrations provide useful information about the severity of inflammation inside the Urinary Bladder. These correlations suggest that CRP concentrations can represent a safe, convenient, and alternative method for evaluating the status of bacterial cystitis [15]. Significantly high CRP values were observed in cases like Lympoma, pyometra, panniculitis, acute pancreatitis, polyarthritis, leptospirosis, babesiosis, parvo viral enteritis, glomerulonephritis, immune mediated disease and malignant neoplasia [16]. Rise in CRP may not be observed in local tumours like leiomyosarcoma, upper respiratory tract infection, diabetes, neurological problems involving intracranial disorders. Since the CRP concentration did not increase in patients with intervertebral disk protrusion, it might be useful in distinguishing arthritis from spinal / brain diseases in patients with lameness. Thus, CRP is a nonspecific inflammatory marker, it could facilitate the diagnosis by indicating the presence and the extent of inflammation

CRP in Elephants

The reference interval for CRP reported herein for Asian elephants (1.3–12.8 mg/l) is like CRP intervals reported in harbor seals (Phoca vitulina) and bottlenose dolphins (Tursiops truncatus). When compare to the CRP, SAA is demonstrated to be the most responsive major APP in elephants [17]. This agrees with previous reports where SAA elevations were noted consistently in elephants with Elephant endotheliotropic herpesvirus (EEHV) and in 2 captive elephants with inflammatory lesions. Further studies are needed to address the reactivity of the CRP reagents with elephant proteins and to consider the use of either elephantspecific reagents or non–antibody-based assays

CRP in Chicken

The birds positive for E.Coli, Pasturella multocida and Staphylococcus aureus infections as well as histomoniasis and adjuvant injection found to be positive for CRP. CRP also positive in clinically normal population. These birds on post-mortem, had lesions consistent with chronic respiratory disease, which is highlighting the use of CRP as a potential biomarker for nonclinical disease [18]. CRP did not rise in chickens as quickly as it does in humans, whereby CRP was detectable 36-48 hours post infection in chickens, compared to 16-18 hours in humans. A more recent study investigated CRP serum concentrations using a human CRP kit and found CRP concentrations to increase

CRP in Equines

A high serum concentration was also found in horses with pneumonia, enteritis, arthritis and after castration. Increased plasma concentration has been observed in carbohydrate induced laminitis. Increased serum concentration of CRP has been found in horses suffering from aseptic inflammation induced by intramuscular turpentine injections [3].

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Abstract

Floating Beds (FB) were prepared in Jaintapur and Kanaighat and one in the Sylhet Agricultural University (SAU) campus of Sylhet region. German and Dhal grass were cultivated infloating beds. There was about 11Kg/sqm and 5.75 Kg/sqm of average fodder production per bed in German and Dhal grass respectively. German grass showed superiority in production and nutritional quality compared to Dhal and local grasses and considered as suitable fodder for FB cultivation. In vitro degradability (IVD) was higher in floating bed German grass than local grasses. By analyzing water quality of the wetland in which fodder bed was constructed it has been revealed that the Dissolved Oxygen (DO) level of the floating bed was at a range in which the fish population had a threat to survive. So, these abandoned ponds were utilized by constructing a floating bed for fodder production. There was a positive correlation between IVD and DO of water. Experimental bucket silage production was carried out for storing German grass and found the potentiality for preserving the grass without any nutrient loss for a long period. Presence of Lactobacillus spp. in silage lowered its pH during ensiled at anaerobic condition and helped to preserve the quality. Confirmation and screening of Lactobacillus spp. in silage was carried out by culturing the microorganisms in a selective De Man, Rogosa & Sharpe (MRS) media followed by different biochemical tests. The FB method of fodder cultivation can be helpful to survive on climate change vulnerability and to ensure sustainable livestock production in haor and low-lying areas.

Keywords: In vitro degradability; Water quality; Bucket silage; Lactobacillus

Abbrevations: LAB: Lactic Acid Bacteria; FBF: Floating Bed Fodder; IVD: In Vitro Degradation; BOD: Biological Oxygen Demand; DO: Dissolved Oxygen; MRS: De Man, Rogosa & Sharpe

Introduction

For sustainable livestock production, climate change acts as a major obstacle in a number of countries. Many countries in the different regions of the world are vulnerable to the impacts of global warming and climate change due to their geographic location, the dominance of floodplains, and low elevation from the sea, high population density, high levels of poverty and overwhelming dependence on nature, its resources and services. Every year different locations are being affected by flood during the rainy season at various locations. The scarcity of green grass increases as the grazing lands and pasture lands are submerged under water during a flood. Many parts of these countries are waterlogged for several months every year during monsoon and the poor peoples of that area suffer a lot mostly to fulfill their various needs. Many of the people have livestock which gives them food, daily income, and financial backup. During the rainy season, their livestock suffers mostly from a lack of food and waterborne diseases. As pasture lands become submerged under water, it limits the livestock to avail green grass. Alternative ways should be developed as sustainable livestock production cannot be achieved by feeding the livestock with low nutrients forages and insufficient feeds during the flood period. These techniques must be inexpensive and easily adopted by farmers so that they can easily produce good quality and sufficient amount of forage that can meet the need in case of flood or another emergency crisis. To ensure food security for flood-prone and water lodged areas, floating bed (FB) vegetable cultivation has been developed [1]. FB is an innovative technique that can be a good tool for ensuring livestock production when flood submerged the pastureland followed by a severe scarcity of grass to feed the livestock. So it is important to develop ideas that can help the farmers to afford quality and sufficient available forage when flood affect the grazing land. Floating bed (FB) for fodder production is a technique that can be a good tool for livestock production when flood submerged the pastureland followed by a severe scarcity of grass to feed the livestock [2]. Floating bed agriculture is a locally adopted production system in southern Bangladesh. Use of such cultivation practice allow the farmers to cultivate crops against the obstacle of a disaster like a flood, also it is cheap, easy and widely accepted by the local farmers and nowadays practiced in many parts of Bangladesh [2,3]. During monsoon, when farmers face the problem of feed shortage for their livestock, high nutritional fodder production on FB can be a good adaptation program. Islam et al. [2] reported thatGerman grass (Echinochloapolystachya) is suitable for FB as it is aquatic or semiaquatic fodder that has also good nutritional value.Dhal grass (Hymenachneamplexicaulis) could be another candidate suitable for FB as it is a wetland grass inhabiting margins of swamps, river floodplains, and drainage canals, mostly in water to about 2 m deep, occasionally extending into water 3-4 m deep. It can be grown for pasture in natural or artificially inundated pond areas [4].

Islam et al. [2] observed the production of German grass on the floatingbed. Water qualityand the seasoncan vary the production and degradability of these grasses that need to be checked. Again, maintaining green fodder availability round the year is a challenge in livestock farming. For proper livestock farming, it is desirable that surplus green grass to be preserved with a minimum loss of nutrients for supply during lean periods when the availability of organic fresh forage is negligible. For forage preservation, silage production may be a key component of high input systems. It has allowed farmers to intensify the productivity of the land and the productivity of the cows independently from each other. As silage making allows storage and preservation of feed resources for months, farmers can focus to maximize the yield of digestible nutrients (energy, protein, etc.), can maximize milk production per cow throughout the year. Fermentation in silage reduces harmful nitrates accumulated in plants during droughts [5]. Therefore, to ensure the feed security of livestock during the rainy season, two upazilaof Sylhet district in Bangladesh namely Kanaighat and Jaintapur were selected. There several floating beds were developed for cultivating fodder (German and Dhal grass) as well as silage production and subsequent research works were carried out for microbial and biochemical assessment of the forage

Materials and Methods

Selection of Study area and Contract Farmers

A survey was performed among the villagers from the different unions under Kanaighat and Jaintapurupazila, Sylhet, Bangladesh. A prepared questionnaire was used to evaluate the socioeconomic status of the villagers. Farmers who have a minimum of five cattle, have a pond nearby and interested to improve their animal management system to develop their cattle stock were selected

Training to the Farmers

A day-long training program was organized at Jingabari and Darbast union Parishad complex of Kanaighat and Jaintapurupazila respectively. All Farmers, selected according to survey, gathered together in the training program.

Preparation of Floating bed

Seven floating beds in Jaintapur, four in Kanaighat and one at Sylhet Agricultural University (SAU) campus, were prepared according to Islam et al. [2]. The size of each floating bed was nearly about 18.5sqm (200 square feet) but the shape of each floating bed was varied with the shape of the pond on which a bed was floated. Material required for a floating bed were bamboo, plastic net, banana plant, soil, cow dung, water hyacinth, rope, and knife. A bamboo frame was prepared and covered with a plastic net. Four pieces of mature banana plants were fixed below the bamboo frame for primary floating management of the bamboo frame. In some beds, empty plastic water bottles were used as an alternativeto the banana trees to float the bed for a long time. Water hyacinths were stocked on the floating bamboo frame with around one feet height to make the first layer of the floating bed. Then the top layer of floating bed about 3 inches was prepared with soil and cow dung.

Fodder Plantation on the Floating bed

German and Dhal grass was found as suitable for FB fodder cultivation [2,6]. Cuttings of German and Dhal grasses were prepared. Each cutting contained three complete internodes with four nodes. The cuttings were planted alternatively on row by row. The distance of one row to another was about 0.25m. German grasses were planted on four floating beds and Dhal grasses were planted on three floating beds at Jaintapur. Among the fourfloating bed with German grass, two were used with a plastic bottle instead of Banana plant. The floating beds at Jaintapur were constructed from July to September when there was flood water available in this area. In Kanaighat only German grass was planted. Among the four-floating bed, two were constructed in October, that is late rainy season and two were constructed in December, i.e. in the winter and dry season to check the production difference in the different season. One floating bed was constructed at SAU campus with German grass in the rainy season.

Care and Management of Floating Bed Fodder Cultivation

There was regular check-up of the bamboo frames that was supporting the structure of the floating bed. The beds were always kept enough away from the pond bank to secure the beds from cattle attack.

Determination of Water Quality of the Floating Bed

The water sample was collected from the ponds where the floating bed was constructed. Two 300ml biochemical oxygen demand (BOD) bottle was filled with the sample water. One bottle was kept for measuring dissolve oxygen (DO) and another one was incubated at room temperature in a dark place for five days. One ml of manganese sulfate was added into the bottle by pipetting. One ml of freshly prepared solution of potassium hydroxide and potassium iodidewas then added. The bottle was shaken to mix the reagents and allowed to stay for five minutes. Light yellow colored precipitate produced to indicate the presence of dissolved oxygen. One ml of concentrated sulphuric acid was added into the BOD bottle and shaken vigorously to mix it well and waited for twenty minutes. After that, the hundred ml of water from BOD bottle was taken into a conical flask and 5-6 drops of the freshly prepared starch solution were added. The solution was then titrated by using 0.025 N sodium thiosulfate drop by drop from the burette. The color changed from blue to coller less indicates the titration point. The initial and final burette reading was then calculated to measure the DO of water. After 5 days, the incubated bottle was then tested by the discussed procedure to measure the DO of the incubated BOD bottle. The difference between the DO of the first day and DO after 5 days indicate the BOD of the water sample [7].

Sample Collection and Fodder Production Evaluation

The first harvest/cut was carried out according to table 1 after plantation. Samples from floating bed among the farmers of Kanaighat and Jaintapurupazila were collected for the study of present research work for production, microbial and nutritional evaluation. Similarly, three local grasses namely Durba, Binna, and Katu, grown naturally in Kanaighat and Jaintapur area, were collected. All the German and Dhal grasses were harvested by cutting at the fourth node (between fourth and fifth internodes) from the base/root. For local grasses, only the Aerial part was collected. Fodders produced on 1sqm space were taken and weighed to evaluate production performance. Spring balance of 20kg was used for this purpose. Then the collected fodders were shifted to Biochemistry laboratory of SAU for nutritional analysis.

Nutritional Evaluation of the Fodders

Sample Preparation

The fodders from the first cut of the floating beds were collected for the analysis. For preparation, the whole grass sample was cut in pieces of less than one cm size with a knife. After taking samples for dry matter and ash test, rest of the samples were dried at 105 °C for overnight, grinded with blender machine and kept separately in an airtight sample bottle. Eleven samples of floating bed from Jaintapur and Kanaighat region, one from SAU campus and three local grasses from the Jaintapur and Kanaighat area, a total of 15 samples were processed. From each of the samples, with three replication cycle, fodders were prepared for proximate analysis and in vitro trial.

Proximate Analysis

The proximate analysis including dry matter (DM), Ash, crude protein (CP), and ether extract (EE) of the fodder samples was performed according to Islam et al. & Khan [2,8].

In vitro Evaluation of the Fodders

Collection of rumen fluid from Cattle Rumen fluid (RF) was collected from healthy slaughtered cattle. The RF was then transported in the insulated flasks under anaerobic conditions to the laboratory that was preheated at 39 °C with water. The RF was strained through a porous cloth into the pre-warmed McDougall buffer at a 1:4 ratio to prepare the inoculums [9]. The flasks were then screw capped and kept at 39 °C in a water bath until used.

Measurement of In Vitro Degradability of Fodders by Rumen Fluid

In Vitro degradability (IVD) was performed according toKhan and Chaudhry [9]. For the 24-hour IVD test, 0.3g of the sample was shifted to a 50ml falcon tube and 30ml of buffered inoculum was poured on it. The tube was screw capped and mixed by updown movement and incubated 24 hours at water bath. Sample from each tube was filtered by a suction pump and the filter paper with residue was dried in an oven and the dry matter was checked and calculated.

Silage Production from Fodder

A technique called ‘Bucket Silage’ was used for the production of silage using German grass collected from a floating bed. A freshly new bucket (25L) was used for storing the silage. About 24kg chopped fodder material was uniformly mixed with one kg molasses

The grass materials were chopped to a short length (1-3cm) and filled into a bucket and sealed tightly to make the bucket airtight to maintain anaerobic condition for fermentation. The bucket was then kept for 28 days for the ensiling the fodder

Screening and Confirmation of Lactic Acid Bacteria (LAB)from Silage

Culturing in MRS Media

Microorganisms from silage were first cultured in nutrients broth and after 24 hours of incubation microbes from the nutrients broth, was cultured in lactobacillus specific MRS media. After 3 days of the inoculation whitish round culture were appeared, these were then subsequently subcultured

Biochemical tests for Confirmation of LAB from Silage

For the confirmation of the presence of Lactobacillus spp. gram Staining, catalase test, oxidase test, indole test, methyl red (MR) test, voges–proskauer (VP) test and carbohydrate fermentation test were performed

Statistical Analysis

Microsoft Excel was used for statistical analysis. Dissolved oxygen (DO) was compared with in vitro degradability (IVD), in data analysis following correlation. Single factor ANOVA was used to measure significant variation among the various samples.

Result

Fodder Production from Floating Bed

The production of fodder from the floating beds significantly (P<0.05) differed from each other and also differed from floating bed in SAU campus (Table 1). Production of floating bed German grass was higher compared to the floating bed Dhal grass and local grasses (Durba, Katu. Binna). The maturity of grass took longer time (90 days) in Kanaighat which were planted in winter. The picture of mature grass in a floating bed is given in Figure 1

Determination of Water Quality

By analyzing water quality of the wetland in which fodder bed was constructed it has been revealed that the DO level of the floating bed was at a range in which the fish population had a threat to survive. So, these abandoned pondswere utilized by constructing a floating bed for fodder production. Water sample of floating bed quality had revealed in Table 2.

Proximate Analysis and in Vitrodegradabilityof Forages

Though the difference was not significant, the DM was lowest in German grass from Jaintapur region than other grasses. After 24 hours, IVD was higher in some of the German grass of floating bed field (FBF) and in the floating bed Sylhet Agricultural University campus (FBS) than local grasses (Table 3). There was a higher value of IVD of silage than German and local grasses (Table 3).

Production of Silage

After 28 days of ensiling in airtight bucket the quality of good silage (Figure 2) was ensured by the characteristics such as the appearance of the silage was greenish brown, absent of mold, there was a smell of lactic and acetic acid (like dahi and vinegar) indicating good quality silage, When the silage was squeezed, the silage breaks slowly into pieces, indicated good quality. In vitro degradability study showed that silage also made the fodder more digestible In vitro degradability study showed that silage also made the fodder more digestible (Table 3).

Screening of Lactobacillus spp.

After 2 days of incubation on MRS media bacterial culture appeared as small, white creamy, colonies (Figure 3) indicating the presence of Lactobacillus spp. The result of the biochemical test (Table 3) confirmed the presence of Lactobacillus spp. grown on De Man, Rogosa & Sharpe media (MRS) agar media

Discussion

The variation in fodder production was due to the dissimilarity in size, care taken by the farmers and seasonal variation at which floating beds were constructed. The floating beds in Kanaighat region were constructed at the beginning of winter which reduced the production comparing to the floating beds at SAU and Jaintapur region. So, to get a better result in fodder production from floating bed, current research suggested that floating bed should be constructed and fodder harvesting should be performed within the rainy season or the summer. To maintain the structural integrity of the floating bed it is suggested that plastic bottles should be used as an alternative to the banana tree to float the bed properly for a prolongedperiod of time since it was found that the banana tree was susceptible to rotting. Local grasses had a slightly higher average DM comparing to the floating bed fodder (P<0.05) (Table 3) as local grasses were grown naturally in soil. DM of German grass that was produced in winter or dry season was higher. Among the FBF there were lower IVD in which fodder was grown and harvested during winter but was a higher IVD value in fodder which was harvested at rainy season (Table 1).

In silage, fodder was fermented and the cellulosic materials slightly breakdown through lactic acid producing bacteria and that makes fodder easily digestible [10]. The present study revealed that there was a positive correlation between IVD and DO of water (r=0.746) that had the floating bed i.e. the fodder sample from floating bed had a less IVD that had a low DO value. So, the fodder sample from FBS had highest IVD among the floating bed samples as it had a maximum DO value. The quality silage can be a good alternative for the storing fodder materials during the lean period to increase feed security for livestock. Proximate analysis study revealed that it had higher nutritional value than normal grasses. Molecular biology techniques can be carried out to identify the specific strain of Lactobacillus present in the silage in future research work. So, it can be used as a probiotic to convert low-quality forage materials into a nutrient rich one [11].

Conclusion

Farmers of Kanaighat and Jaintapur were very much interested after watching the innovative technique of fodder production in floating bed. It was a completely new practice for them. As this technique didn’t require any chemical fertilizers and construction required only locally available materials it was very much cost effective and easy to develop for the poor farmers. So, farmers can adopt floating bed for producing fodder even in the flooded period and silage during the lean period to preserve feeds as a tool for sustainable livestock production. Future research can be carried out on the influence of these floating bed fodders on growth and milk production performance in the ruminant

Acknowledgment

Acknowledgment to National Agricultural Technology Transfer Program-Phase II Project (NATP-2), Project Implementation Unit (PIU), Bangladesh Agricultural Research Council (BARC) for funding the research

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