*5.1.2 Processing facilities*

In the recent years, a sizeable number of milk processing plants is in operations. These plants pollute the surrounding environment and contribute to climate change by adding solid waste, liquid waste, soil pollution and noise, and air emissions. Of these, wastewater from washing and cleaning operations is the greatest pollutant, and is estimated to be between 5.5 and 30 cubic meter for every cubic meter of milk processed at a typical dairy unit [18, 19].

### **5.2 Awareness and understanding of environmental consequences**

There is a lack of understanding for environmental consequences of livestock in developing countries and the situation in Pakistan is no different, as demonstrated by the Livestock Policy's (2007) failure to consider environment. Awareness and understanding of the environmental consequences of dairying are extremely limited in almost all sectors, especially the government sector and small-scale farming. The focus is on the more obvious contamination, such as flies and odor, rather than the serious threats of land degradation, water pollution, biodiversity erosion, and global climate change.


**14. Pharmacological treatment of neuropathic pain**

Treating neuropathic pain requires a multifaceted approach that aims to eliminate the underlying etiology, when possible, and manage the associated discomforts and emotional distress. Although in some cases it is possible to directly treat the cause of neuropathic pain, for example surgery to alleviate a constricted nerve, it is more likely that the primary cause is untreatable, as is the case with singular traumatic events such as stroke and spinal cord injury and diseases like diabetes. When this is the case, symptom management and pain reduction become the primary focus. Unfortunately, in most cases complete elimination of pain is not a feasible endpoint; a pain reduction of 30% is considered to be efficacious [21]. Additionally, many pharmacological treatments require careful titration and tapering to prevent adverse effects and toxicity. This process may take several weeks to months, and ultimately the drug may be ineffective, necessitating another trial with a different medication. It is therefore necessary that both doctor and patient begin treatment with realistic expectations and goals.

First-line medications for the treatment of neuropathic pain are those that have proven efficacy in randomized clinical trials (RCTs) and are consistent with pooled clinical observations [81]. These include antidepressants, calcium channel ligands, and topical lidocaine [15]. Tricyclic antidepressants (TCAs) have demonstrated efficacy in treating neuropathic pain with positive results in RCTs for central post-stroke pain, PHN, painful diabetic and non-diabetic polyneur‐ opathy, and post-mastectomy pain syndrome [82]. However they do not seem to be effective in treating painful HIV-neuropathy or CIPN [82]. Duloxetine and venlafaxine, two selective serotonin norepinephrine reuptake inhibitors (SSNRIs), have been found to be effective in DPN and both DPN and painful polyneuropathies, respectively [81]. Adverse affects associated with TCAs and SSNRIs are relatively mild and can be mitigated by a slow titration beginning with

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Gabapentin and pregabalin have also demonstrated efficacy in several neuropathic pain conditions including DPN and PHN [81, 82]. Both drugs exert their effects by inhibiting neurotransmitter release through binding of the α2-δ subunit of presynaptic calcium channels [83]. Adverse effects and efficacy of gabapentin and pregabalin are similar; however prega‐ balin may provide more rapid analgesia due to straightforward dosing determined by linear pharmacokinetic [78]. Topical lidocaine (5% patch or gel) has significantly reduced allodynia associated with PHN and other neuropathic pain syndromes in several RCTs [81, 82]. With no reported systemic adverse effects and mild skin irritation as the only concern, lidocaine is an

In the event that first line medications, alone or in combination, are not effective at achieving adequate pain relief, second line medications may be considered. These include opioid analgesics and tramadol, pharmaceuticals which have proven efficacy in RCTs but are associated with significant adverse effects that warrant cautious prescription [15]. Although opioid analgesics are effective pain relievers in several types of neuropathic pain [81, 82, 84], they are associated with misuse or abuse, hypogonadism, constipation, nausea, and immu‐ nological changes [15]. Because many of these side effects can be mitigated by a low dose, careful titration, and short term use, opiates are an appropriate choice for treating acute or episodic neuropathic pain [81]. Careful consideration should be given when prescribing opiates to patients who have a personal or family history of drug or alcohol abuse, and

Tramadol, a weak opioid µ-receptor agonist and serotonin and norepinephrine reuptake inhibitor (SNRI), is more effective than placebo but less effective than strong opioid µ-receptor agonists (e.g. morphine and oxycodone) in treating neuropathic pain [82]. Although the risk is considerably less than opioid analgesics, tramadol is also associated with abuse [81]. A rare but potentially fatal serotonin syndrome has been described, and tramadol may increase the

Recent clinical trials have considered additional intervention strategies with possible utility in treatingneuropathicpain,althoughtheir efficacyremains tobedetermined.Treatments include botulinumtoxinforPHNandpostoperativeallodynia[85,86],highconcentrationcapsaisinpatch forthetreatmentofPHNandpainfulHIVneuropathy[15],andlacosamide,anantiepilepticdrug with suggested efficacy in treating DPN [87-89]. There is also accumulating evidence that

appropriate choice for treating localized peripheral neuropathic pain.

additional monitoring to ensure appropriate use may be necessary.

likelihood of seizures or interact with other medications [15].

a low dose [81].

**Table 1.** A list of bedside tests used to identify signs and symptoms that are suggestive of neuropathic pain.

Recently, the Neuropathic Pain Special Interest Group (NeuPSIG) of the International Asso‐ ciation for the Study of Pain reviewed the evidence–based guidelines for the pharmacological treatment of neuropathic pain and made recommendations that take into account clinical efficacy, adverse effects, effects on health related quality of life, convenience, and cost [81]. These findings as well as more recent evidence are reviewed here.

First-line medications for the treatment of neuropathic pain are those that have proven efficacy in randomized clinical trials (RCTs) and are consistent with pooled clinical observations [81]. These include antidepressants, calcium channel ligands, and topical lidocaine [15]. Tricyclic antidepressants (TCAs) have demonstrated efficacy in treating neuropathic pain with positive results in RCTs for central post-stroke pain, PHN, painful diabetic and non-diabetic polyneur‐ opathy, and post-mastectomy pain syndrome [82]. However they do not seem to be effective in treating painful HIV-neuropathy or CIPN [82]. Duloxetine and venlafaxine, two selective serotonin norepinephrine reuptake inhibitors (SSNRIs), have been found to be effective in DPN and both DPN and painful polyneuropathies, respectively [81]. Adverse affects associated with TCAs and SSNRIs are relatively mild and can be mitigated by a slow titration beginning with a low dose [81].

for example surgery to alleviate a constricted nerve, it is more likely that the primary cause is untreatable, as is the case with singular traumatic events such as stroke and spinal cord injury and diseases like diabetes. When this is the case, symptom management and pain reduction become the primary focus. Unfortunately, in most cases complete elimination of pain is not a feasible endpoint; a pain reduction of 30% is considered to be efficacious [21]. Additionally, many pharmacological treatments require careful titration and tapering to prevent adverse effects and toxicity. This process may take several weeks to months, and ultimately the drug may be ineffective, necessitating another trial with a different medication. It is therefore necessary that both doctor and patient begin treatment with realistic expectations and goals.

14 Peripheral Neuropathy - A New Insight into the Mechanism, Evaluation and Management of a Complex Disorder

**Signs and Symptoms Bedside Test Pathological Response**

Reduced sensation

Sharp burning superficial pain in putative lesion territory as well as

Sharp burning superficial pain in putative lesion territory as well as

as well as unaffected area

unaffected area

unaffected area

intervals for 30s Sharp pain with increasing intensity

Touch skin with cotton swab or

gauze

Paraesthesia Reported – grade intensity 1-10

Shooting Reported – grade intensity 1-10 Ongoing Reported – grade intensity 1-10

Dynamic Mechanical Move object (cotton swab or gauze)

Punctate Mechanical Pinprick with sharp object

Temporal Summation Pinprick with sharp object at 3s

These findings as well as more recent evidence are reviewed here.

along skin

Hypoalgesia Prick skin with pin Reduced sensation

Cold Touch skin object <20°C Painful, burning sensation Heat Touch skin object "/>40°C Painful, burning sensation

Static Mechanical Apply gentle pressure to skin Dull pain in putative lesion territory

Recently, the Neuropathic Pain Special Interest Group (NeuPSIG) of the International Asso‐ ciation for the Study of Pain reviewed the evidence–based guidelines for the pharmacological treatment of neuropathic pain and made recommendations that take into account clinical efficacy, adverse effects, effects on health related quality of life, convenience, and cost [81].

**Table 1.** A list of bedside tests used to identify signs and symptoms that are suggestive of neuropathic pain.

*Abnormal Sensations*

Hypoesthesia

*Spontaneous Pain*

Allodynia/Hyperalgesia

*Evoked Pain*

Gabapentin and pregabalin have also demonstrated efficacy in several neuropathic pain conditions including DPN and PHN [81, 82]. Both drugs exert their effects by inhibiting neurotransmitter release through binding of the α2-δ subunit of presynaptic calcium channels [83]. Adverse effects and efficacy of gabapentin and pregabalin are similar; however prega‐ balin may provide more rapid analgesia due to straightforward dosing determined by linear pharmacokinetic [78]. Topical lidocaine (5% patch or gel) has significantly reduced allodynia associated with PHN and other neuropathic pain syndromes in several RCTs [81, 82]. With no reported systemic adverse effects and mild skin irritation as the only concern, lidocaine is an appropriate choice for treating localized peripheral neuropathic pain.

In the event that first line medications, alone or in combination, are not effective at achieving adequate pain relief, second line medications may be considered. These include opioid analgesics and tramadol, pharmaceuticals which have proven efficacy in RCTs but are associated with significant adverse effects that warrant cautious prescription [15]. Although opioid analgesics are effective pain relievers in several types of neuropathic pain [81, 82, 84], they are associated with misuse or abuse, hypogonadism, constipation, nausea, and immu‐ nological changes [15]. Because many of these side effects can be mitigated by a low dose, careful titration, and short term use, opiates are an appropriate choice for treating acute or episodic neuropathic pain [81]. Careful consideration should be given when prescribing opiates to patients who have a personal or family history of drug or alcohol abuse, and additional monitoring to ensure appropriate use may be necessary.

Tramadol, a weak opioid µ-receptor agonist and serotonin and norepinephrine reuptake inhibitor (SNRI), is more effective than placebo but less effective than strong opioid µ-receptor agonists (e.g. morphine and oxycodone) in treating neuropathic pain [82]. Although the risk is considerably less than opioid analgesics, tramadol is also associated with abuse [81]. A rare but potentially fatal serotonin syndrome has been described, and tramadol may increase the likelihood of seizures or interact with other medications [15].

Recent clinical trials have considered additional intervention strategies with possible utility in treatingneuropathicpain,althoughtheir efficacyremains tobedetermined.Treatments include botulinumtoxinforPHNandpostoperativeallodynia[85,86],highconcentrationcapsaisinpatch forthetreatmentofPHNandpainfulHIVneuropathy[15],andlacosamide,anantiepilepticdrug with suggested efficacy in treating DPN [87-89]. There is also accumulating evidence that intravenous Ca2+ and Mg2+ may be effective at preventing CIPN caused a commonly used chemotherapeutic, oxaliplatin, without attenuating its antineoplastic efficacy [9].

of patients who experience similar "symptom constellations" representing distinct pathophy‐ siological mechanisms [95]. Furthermore, these symptom constellations can be seen, albeit in different proportions, across neuropathic pain syndromes, suggesting that the same underly‐ ing mechanism can cause neuropathic pain within and apart from the initiating etiology. Hypothetically, with this understanding comes an approach of targeted treatment that aims to identify the pathophysiological mechanism and specifically inhibit, block, or enhance the offending molecules. To implement this type of treatment will require a more intimate understanding of the mechanisms of neuropathic pain and the corresponding symptom manifestations. As this becomes defined, specific treatments can begin to emerge, and clinical

**Signs and Symptoms Example Mechanisms Targeted Treatment**

Inflammation in nerve root, central sensitization (potentiation),

Modulation of TRPM8 or Na2+ channels in peripheral nociceptors

and facilitation), disinhibition

and facilitation), disinhibition

**Table 2.** Hypothetical examples of how signs and symptoms obtained in a bedside examination might indicate underlying pathophysiological mechanism. Once a putative mechanism has been established there is a potential for

Modulation of unknown mechanoreceptors in peripheral

nociceptors, TRPA1

selective and specifically targeted treatments to be applied. For a comprehensive review see [21].

Selective Na2+ channel blocker

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Cytokine antagonists, Calcium channel blocker, NMDA receptor

TRPM8 receptor antagonist, Selective Na2+ channel blocker

Ca2+ channel blocker, NMDA receptor antagonist

Ca2+ channel blocker, NMDA receptor antagonist

Ca2+ channel blocker, NMDA receptor antagonist

antagonist

nociceptors TRPV1 receptor antagonist

?

channel dysregulation

disinhibition

trials can test the efficacy of this approach. See Table 2 for examples.

Shooting Ectopic impulse generation, Na2+

Heat Modulation of TRPV1 in peripheral

Dynamic Mechanical Central sensitization (potentiation

Punctate Mechanical Central sensitization (potentiation

Temporal Summation Central sensitization

*Spontaneous Pain*

Ongoing

*Evoked Pain*

Cold

Allodynia/Hyperalgesia

Static Mechanical

### **15. Non-pharmacological treatment of neuropathic pain**

The use of alternative and complementary medicine is on the rise, particularly in the United States [90]. Although anecdotal evidence abounds, there are relatively few RCTs supporting the use of such therapies. It is important in considering these treatments, however, that the lack of evidence is not read as evidence of lacking efficacy. The scarcity of well controlled, robust clinical trials considering non-pharmacological treatments of chronic pain makes it difficult to recommend or dismiss these alternative treatments. A few studies have examined the use of acupuncture, herbal therapy, massage, hypnosis, and biofeedback on easing chronic pain but have yielded mixed results (for a review see [90]). The difficulty in standardizing treatment, inherent to these multi-faceted approaches, is a major obstacle in drawing reliable conclusions. Additionally, small sample sizes and lack of obvious controls are also significant barriers. Despite these hurdles, which obscure evidence-based conclusions, non-pharmaco‐ logical treatments are often prescribed in conjunction with evidence-based recommendations due to low risk of accompanying adverse effects.

Deep brain stimulation, a neurosurgical technique by which an implanted electrode delivers controlled electrical impulses to targeted brain regions, has demonstrated some efficacy in treating chronic pain but is not routinely employed due to a high risk-to-benefit ratio [91]. Targeting the periventricular/periaqueductal gray, internal capsule, and sensory thalamus has demonstrated efficacy in various pain conditions [91], but not all types of chronic pain are responsive. An intriguing new target, the NAc, has recently emerged as a potential site for deep brain stimulation as it has demonstrated efficacy in a case study of post-stroke pain [92]. As studies of pain processing in the brain have suggested, the pattern of activity in the NAc is divergent in nociceptive and chronic pain representation, validating this structure as a possible therapeutic target [69].

Another type of electro-stimulation device is emerging as a promising therapeutic tool for the treatment of neuropathic pain [93, 94]. Delivering repeated pulses of electrical stimulation trans-cutaneously, termed Scrambler therapy, has demonstrated some efficacy with lasting effects in CIPN [94], postsurgical pain, PHN, and spinal canal stenosis [93]. With few adverse effects and low associated risk, this may be a viable alternative to pharmacological treatment.


**3.3. Peach**

For peach, the use of Ψstem for defining thresholds under DI conditions is referred by Girona et al. [70], who found the value −1.5 MPa, the limit over which the impairing of bloom fertility appears. Naor et al. [39] have observed that the value of −2.0 MPa for SWP was a threshold for the occurrence of double fruits, while Lopez et al. [71] suggest a threshold of −1.05 MPa to obtain fruits with positive effects on consumer acceptance, without significant impacts on fruit composition and yield, as they have observed that a threshold of −1.25 MPa would reduce fruit size and yield, even if advantageous for consumer acceptance.

Other authors, using relative transpiration (RT), have observed that a minimum value of 0.7 has to be observed to avoid yield and quality losses [72].

Using the relationship between (RT) and Ψpd, it was observed [73] that the Ψpd threshold corresponding to RT equal to 0.7 is −0.33 MPa. Using CWSI, based on the temperature differences between canopy and air, a threshold of 0.5 was found to trigger irrigation [74]. It was also found that it is possible to identify a threshold in the relationship between gs and Ψp, corresponding to a change in the plant behavior, equal to Ψpd = −0.45 MPa [75].

information is quite complex and scattered. Classical recommendations often include the use of leaf water potential [94]; a new water stress index based on a water balance model was proposed and tested by Gaudin et al. [95] as a tool for classifying water stress experienced by

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Shoots growth and fruits development are cyclical and both are repeated on an annual basis, but only vegetative growth is completed in the same year, while olives production needs two consecutive seasons [96]. In the first one, the formation of the buds and their floral induction take place. In the following year, flower development occurs as well as flowering, fruit set, growth, and oil accumulation. In Mediterranean climate conditions of northern hemisphere, shoot growth takes place from March until the middle of July, although a second flow of growth can occur in late August, when olive trees are fully irrigated, or at the beginning of autumn rainfall [97]. Water deficit reduces shoots growth and has a negative effect on the potential production of the following year. Flowering occurs at the end of spring, and it is very sensitive to water deficit [63], or at high temperatures. Fruit set is very sensitive to water deficit and fruit growth has a double sigmoid behavior [96, 98] with three main stages, as follows. Phase I is the fast-growing, when both the cell division and expansion contribute to the size increase, the endocarp being the main tissue in development, reaching 80% of the volume of the olives [98] with full expansion about 8 weeks after full bloom [99]. The occurrence of water deficit in this stage results in a small endocarp and extreme water stress can compromise the viability of the fruit. Phase II, of slow-growth, is less sensitive to water deficit [100], when the endocarp progressively hardens and both the embryo and the endocarp reach their final size [98]. During phase III, of fast growing, parenchyma cells of the mesocarp experience a large increase in size, entirely due to cell expansion, and the oil biosynthesis begins [98]; so water availability for the fruit determines its size and the accumulation of oil. Thus, water deficit may produce small fruits and the mesocarp/endocarp ratio is reduced due to decreased weight of the mesocarp.

Many studies had showed that high soil water availability increments yield components such as fruit number, fruit fresh weight, fruit volume, pulp:stone ratio, and oil content; therefore, increasing fruit and oil yields [12, 63] and that water scarcity can have a negative effect, depending on its level. In addition, irrigation regime can influence the relationship between

Hernandez-Santana [102] observed that olive trees prioritize fruit growth and oil content accumulation over vegetative growth, suggesting a higher sink strength for reproductive growth

**4. Responses to deficit irrigation regarding agronomic aspects and**

grapevines in vineyards.

*4.1.1. Vegetative growth and production cycle*

*4.1.2. Olive response to water deficit*

vegetative and reproductive growth [101].

**quality**

**4.1. Olive**


*2.1. LIN28 Knockdown in Trophectoderm Resulted in Reduced Proliferation of Trophoblast Cells and Lower Expression of Proliferation-Associated Genes*

Day 9-hatched blastocysts were infected with lentiviral particles expressing shRNA to knockdown LIN28A (AKD) or LIN28B (BKD), or scramble control shRNA (SC). The blastocysts treated with lentiviral particles were surgically transferred to synchronized ewes at day 9 of estrus. The conceptuses were collected at day 16 of gestation and trophectoderm (TE) was separated from embryo. LIN28A and LIN28B mRNAs and proteins were quantified by real-time PCR and Western blot. LIN28A mRNA and protein was significantly reduced in AKD TE while LIN28B mRNA and protein was significantly reduced in BKD TE compared to SC (Figure 1A,B). As expected, due to reduced LIN28, *let-7* miRNAs (*let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i*) were significantly higher in AKD and BKD TE compared to SC, and there was no significant change in *let-7* miRNAs between AKD and BKD TE (Figure 1C). These results suggest that reduced LIN28A or LIN28B led to significant increase in *let-7* miRNAs.

**Figure 1.** LIN28A or LIN28B knockdown and *let-7* miRNAs in day 16 sheep TE. (**A**) *LIN28A* and *LIN28B* mRNA in AKD (*n* = 5) and BKD (*n* = 6) day 16 TE compared to SC (*n* = 6). (**B**) Representative immunoblots for LIN28A, LIN28B, and β-actin in AKD, BKD, and SC day 16 TE, and densitometric analysis. (**C**) *Let-7* miRNAs in AKD and BKD day 16 TE and SC. \* *p* < 0.05 vs. SC.

To determine the effect of LIN28 knockdown on conceptus elongation, we measured the length of TE. Knockdown of LIN28A or LIN28B resulted in a significant reduction in day 16 TE length compared to SC (Figure 2A,B). There was no significant difference in elongation of AKD vs. BKD TE. This data suggest knockdown of either LIN28A or LIN28B in vivo resulted in reduced proliferation of trophoblast cells.

**Figure 2.** (**A**) Conceptus length at day 16 after LIN28A knockdown (AKD, *n* = 6) and LIN28B knockdown (BKD, *n* = 8) compared to scramble control (SC, *n* = 5). (**B**) Representative images of day 16 sheep conceptuses for AKD, BKD, and SC day 16 TE; \* *p* < 0.05 vs. SC.

Due to the potential for reduced proliferation of trophoblast cells in the AKD and BKD conceptuses, we measured the mRNA and protein levels of *let-7*-regulated proliferation-associated genes. The mRNA and protein levels of IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, and c-MYC were significantly reduced in AKD and BKD day 16 TE compared to SC (Figure 3, Figure 4). These results suggest that high *let-7* miRNAs in AKD and BKD TE led to a significant reduction in expression of IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, and c-MYC, and the reduced proliferation of trophoblast cells in AKD and BKD TE is due to significantly reduced expression of these proliferation-associated genes.

**Figure 3.** *IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B,* and *c-MYC* mRNA in AKD and BKD day 16 TE compared to SC (*n* = 5), \* *p* < 0.05 vs. SC.

**Figure 4.** Representative immunoblots for IGF2BP1, IGF2BP2, IGF2BP3, HMGA1, ARID3B, c-MYC, and β-actin, and densitometric analysis of immunoblotting results in AKD and BKD day 16 sheep TE compared to SC (*n* = 3), \* *p* < 0.05 vs. SC.